Anti-VEGF antibodies and their uses

ABSTRACT

The present disclosure relates to antibodies directed to vascular endothelial growth factor (“VEGF”) and uses of such antibodies, for example to treat diseases associated with the activity and/or overproduction of VEGF.

1. CROSS-REFERENCE TO RELATED APPLICATIONS AND SEQUENCE LISTING

This application claims the benefit under 35 U.S.C. §119(e) of U.S.provisional application No. 61/218,005, filed Jun. 17, 2009, thecontents of which are incorporated herein by reference in theirentireties.

The instant application contains a Sequence Listing which has beensubmitted via EFS-Web and is hereby incorporated by reference in itsentirety. Said ASCII copy, created on Jun. 15, 2010, is named381493US.txt and is 132,486 bytes in size.

2. FIELD OF THE INVENTION

The present invention relates to anti-VEGF antibodies, pharmaceuticalcompositions comprising anti-VEGF antibodies, and therapeutic uses ofsuch antibodies.

3. BACKGROUND

Angiogenesis has emerged as attractive therapeutic target due to itsimplication in a variety of pathological conditions, including tumorgrowth, proliferative retinopathies, age-related macular degeneration,rheumatoid arthritis (RA), and psoriasis (Folkman et al., 1992, J. Biol.Chem. 267:10931-10934). The first indication of specific molecularangiogenic factors was based on the observation of the strongneovascular response induced by transplanted tumors. It is now knownthat angiogenesis is essential for the growth of most primary tumors andtheir subsequent metastasis. Numerous molecules have since beenassociated with the positive regulation of angiogenesis, includingtransforming growth factor (TGF)-α, TGF-β, hepatocyte growth factor(HGF), tumor necrosis factor-α, angiogenin, interleukin (IL)-8, andvascular endothelial growth factor (VEGF, also referred to as VEGFA orvascular permeability factor (VPF)) (Ferrara et al., 2003, NatureMedicine 9:669-676).

The VEGF proteins are important signaling proteins involved in bothnormal embryonic vasculogenesis (the de novo formation of the embryoniccirculatory system) and abnormal angiogenesis (the growth of bloodvessels from pre-existing vasculature) (Ferrara et al., 1996, Nature380:439-442; Dvorak et al., 1995, Am. J. Pathol. 146:1029-1039). VEGF isassociated with solid tumors and hematologic malignancies, interocularneovascular syndromes, inflammation and brain edema, and pathology ofthe female reproductive tract (Ferrara et al., 2003, Nature Medicine9:669-676). VEGF mRNA is over-expressed in many human tumors, includingthose of the lung, breast, gastrointestinal tract, kidney, pancreas, andovary (Berkman et al., 1993, J. Clin. Invest. 91:153-159). Increases inVEGF in the aqueous and vitreous humor of the eyes have been associatedwith various retinopathies (Aiello et al., 1994, N. Engl. J. Med.331:1480-1487). Age-related macular degeneration (AMD), a major cause ofvision loss in the elderly is due to neovascularization and vascularleakage. The localization of VEGF in the choroidal neovascular membranesin patients affected by AMD has been shown (Lopez et al., 1996, Invest.Ophtalmo. Vis. Sci. 37:855-868).

The VEGF gene family includes the prototypical member VEGFA, as well asVEGFB, VEGFC, VEGFD, and placental growth factor (PLGF). The human VEGFAgene is organized as eight exons separated by seven introns. At leastsix different isoforms of VEGF exist, VEGF₁₂₁, VEGF₁₄₅, VEGF₁₆₂,VEGF₁₆₅, VEGF_(165b), VEGF₁₈₃, VEGF₁₈₉, and VEGF₂₀₆, where thesubscripts refer to the number of amino acids remaining after signalcleavage. Native VEGF is a 45 kDa homodimeric heparin-bindingglycoprotein (Ferrara et al., 2003, Nature Medicine 9:669-676). VEGF(specifically VEGFA) binds to two related receptor tyrosine kinases,VEGFR-1 (also referred to as Flt-1) and VEGFR-2 (also referred to asFlk-1 or kinase domain region (KDR) or CD309). Each receptor has sevenextracellular and one transmembrane region. VEGF also binds to theneuropilins NRP1 (also referred to as vascular endothelial cell growthfactor 165 receptor (VEGF165R) or CD304) and NRP2 also referred to asvascular endothelial cell growth factor 165 receptor 2 (VEGF165R2)).

Given its central role in regulating angiogenesis, VEGF provides anattractive target for therapeutic intervention. Indeed, a variety oftherapeutic strategies aimed at blocking VEGF or its receptor signalingsystem are currently being developed for the treatment of neoplasticdiseases. The anti-VEGF antibody bevacizumab, also referred to as rhuMAbVEGF or Avastin®, is a recombinant humanized anti-VEGF monoclonalantibody created and marketed by Genentech (Presta et al., 1997, CancerRes. 57:4593-4599). In order to construct bevacizumab thecomplementarity-determining regions (CDRs) of the murine anti-VEGFmonoclonal antibody A.4.6.1 were grafted onto human frameworks and anIgG constant region. Additional mutations outside the CDRs were thenintroduced into the molecule to improve binding, affording an antibodyin which ˜93% of the amino acid sequence is derived from human IgG₁ and˜7% of the sequence is derived from the murine antibody A.4.6.1.Bevacizumab has a molecular mass of about 149,000 Daltons and isglycosylated.

Ranibizumab is an affinity maturated Fab fragment derived frombevacizumab. Ranibizumab has a higher affinity for VEGF and also issmaller in size, allowing it to better penetrate the retina, and thustreat the ocular neovascularization associated with AMD (Lien andLowman, In: Chemajovsky, 2008, Therapeutic Antibodies. Handbook ofExperimental Pharmacology 181, Springer-Verlag, Berlin Heidelberg131-150). Ranibizumab was developed and is marketed by Genentech underthe trade name Lucentis®.

Treatment of cancer patients with a regimen that includes Avastin® canresult in side effects including hypertension, proteinuria,thromboembolic events, bleeding and cardiac toxicity (Blowers & Hall,2009, Br. J. Nurs. 18(6):351-6, 358). Also, despite being a humanizedantibody, bevacizumab can elicit an immune response when administered tohumans. Such an immune response may result in an immune complex-mediatedclearance of the antibodies or fragments from the circulation, and makerepeated administration unsuitable for therapy, thereby reducing thetherapeutic benefit to the patient and limiting the re-administration ofthe antibody.

Accordingly, there is a need to provide improved anti-VEGF antibodies orfragments that overcome one or more of these problems, for example, bygenerating variants with higher affinity than bevacizumab that can beadministered at reduced dosages, or variants with reduced immunogenicityand other side-effects as compared to bevacizumab.

Citation or identification of any reference in Section 3 or in any othersection of this application shall not be construed as an admission thatsuch reference is available as prior art to the present disclosure.

4. SUMMARY

The present disclosure relates to variants of the anti-VEGF antibodybevacizumab with reduced immunogenicity and/or improved affinity towardsVEGF as compared to bevacizumab or ranibizumab. Bevacizumab has threeheavy chain CDRs, referred to herein (in amino- to carboxy-terminalorder) as CDR-H1, CDR-H2, and CDR-H3, and three light chain CDRs,referred to herein (in amino- to carboxy-terminal order) as CDR-L1,CDR-L2, and CDR-L3. The sequences of the bevacizumab CDRs are shown inFIGS. 1A and 1B, and their numbering is set forth in FIG. 5 (for heavychain CDRs) and FIG. 6 (for light chain CDRs). A related antibody,ranibizumab, was generated by affinity maturation of bevacizumab.Ranibizumab has identical CDR-L1, CDR-L2, CDR-L3 and CDR-H2 sequences tobevacizumab, but varies in its CDR-H1 and CDR-H3 sequences from those ofbevacizumab. The heavy and light chain sequences of ranibizumab areshown in FIG. 1C, and the CDRs are set forth in FIG. 1D.

The antibodies of the disclosure generally have at least one amino acidsubstitution in at least one heavy chain CDR as compared to bevacizumaband ranibizumab.

In certain aspects, the anti-VEGF antibodies include at least onesubstitution as compared to bevacizumab or ranibizumab selected fromN31F in CDR-H1; K64S in CDR-H2; K64Q in CDR-H2; Y53F in CDR-H2; H97E inCDR-H3; H97D in CDR-H3; H97P in CDR-H3; Y98F in CDR-H3; Y99E in CDR-H3;Y99D in CDR-H3; S100aG in CDR-H3, and T51A in CDR-L2. In other aspects,the anti-VEGF antibodies include at least one substitution selected fromFIGS. 12 and 13. Additional mutations that can be incorporated into theimproved affinity variant antibodies can be candidate deimmunizingsubstitutions, such as those described in FIG. 10, as well as othermutations, e.g., substitutions, that do not destroy the ability of theantibodies to bind to VEGF, including but not limited to the mutationsdescribed in FIGS. 14 and 15, or known mutations, such as the mutationsdescribed in FIGS. 16A-16I and 17. Yet further mutations that can beincorporated include but are not limited to the mutations described inFIGS. 18-20.

In specific embodiments, the anti-VEGF antibodies of the disclosureinclude a combination of substitutions selected from FIG. 11, andoptionally one or more additional mutations, e.g., candidatedeimmunizing substitutions, such as those described in FIG. 10, as wellas other mutations, e.g., substitutions, that do not destroy the abilityof the antibodies to bind to VEGF, including but not limited to themutations described in FIGS. 14 and 15, or known mutations, such as themutations described in FIGS. 16A-16I and 17. Yet further mutations thatcan be incorporated into the anti-VEGF antibodies of the disclosureinclude but are not limited to the mutations described in FIGS. 18-20.

In other embodiments, the anti-VEGF antibodies of the disclosure includeone or more of the following CDR substitutions: K64S (CDR-H2), K64Q(CDR-H2), Y53F and K64Q (CDR-H2), H97E and Y98F (CDR-H3), or T51A(CDR-L2). The anti-VEGF antibodies can also optionally include one ormore additional mutations or combinations of mutations selected from oneor more of FIGS. 10, 11, 12, 13, 14, 15, 16A to 16I, or 17-20.

Further CDR substitutions can include N31F (CDR-H1), H97E (CDR-H3), H97D(CDR-H3), H97P (CDR-H3), Y99E (CDR-H3), Y99D (CDR-H3), S100aG (CDR-H3)wherein position 3 in CDR-H3 optionally is not tyrosine, T28P, N31F,N31G and N31M (CDR-H1), H97A, H97Q, H97S, H97T, S100aD, S100aE, andS100Av (CDR-H3), T30W, T30R or T30Q (CDR-H1), Y53F, T58F, A61G, A61K,A61R, A61H, A61Y, K64G, K64E, R65L, R65T, R65A, R65E, and R65D (CDR-H2),and Y98F and Y100eF (CDR-H3). The CDRs optionally contain one or moreadditional mutations or combinations of mutations selected from one ormore of FIGS. 10, 11, 12, 13, 14, 15, 16A to 16I, and 17.

Yet further substitutions can include heavy chain CDR substitutionsincluding a combination of substitutions selected from: (a) N31F inCDR-H1, H97D in CDR-H3, Y99D in CDR-H3, and S100aG in CDR-H3; (b) N31Fin CDR-H1, H97P in CDR-H3, Y99D in CDR-H3, and S100aG in CDR-H3; (c)N31F in CDR-H1, H97P in CDR-H3, and Y99E in CDR-H3; (d) N31F in CDR-H1,H97E in CDR-H3, and Y99E in CDR-H3; (e) N31F in CDR-H1, H97D in CDR-H3,and Y99E in CDR-H3; (f) N31F in CDR-H1, H97E in CDR-H3, Y99D in CDR-H3,and S100aG in CDR-H3; (g) N31F in CDR-H1, Y99D in CDR-H3, and S100aG inCDR-H3; (h) N31F in CDR-H1, H97P in CDR-H3, and Y99D in CDR-H3; (i) N31Fin CDR-H1, H97D in CDR-H3, and S100aG in CDR-H3; (j) N31F in CDR-H1 andS100aG in CDR-H3; or (k) N31F in CDR-H1, H97P in CDR-H3, and S100aG inCDR-H3. Further optional substitutions can include one or moreadditional mutations or combinations of mutations selected from one ormore of FIGS. 11, 12, 13, 14, 15, 16A to 16I, and 17.

Still further heavy chain substitutions can include at least onesubstitution selected from A61F in CDR-H2, A61E in CDR-H2, A61D inCDR-H2, D62L in CDR-H2, D62G in CDR-H2, D62Q in CDR-H2, D62T in CDR-H2,D62K in CDR-H2, D62R in CDR-H2, D62E in CDR-H2, D62H in CDR-H2, K64S inCDR-H2, K64V in CDR-H2, K64Q in CDR-H2, R65V in CDR-H2, R65F in CDR-H2,R65H in CDR-H2, R65N in CDR-H2, R65S in CDR-H2, R65Q in CDR-H2, R65K inCDR-H2, R65I in CDR-H2, and Y98H in CDR-H3. Optionally, one or moreadditional mutations or combinations of mutations can be included asselected from one or more of FIGS. 11, 12, 13, 14, 15, 16A to 16I, and17.

In certain aspects, the antibodies of the disclosure have VH and VLsequences having at least 80% sequence identity (and in certainembodiments, at least 85%, at least 90%, at least 95%, at least 98%, orat least 99% sequence identity) to the VH and VL sequences ofbevacizumab or ranibizumab, and include at least one amino acidsubstitution in at least one CDR as compared to bevacizumab orranibizumab. In other aspects, the antibodies of the disclosure have VHand VL sequences having at least 80% sequence identity (and in certainembodiments, at least 85%, at least 90%, at least 95%, at least 98%, orat least 99% sequence identity) to the VH and VL sequences ofbevacizumab or ranibizumab, and include at least one amino acidsubstitution in at least one framework region as compared to bevacizumabor ranibizumab. In specific embodiments, the percentage sequenceidentity for the heavy chain and the light chain compared to the VH andVL sequences of bevacizumab or ranibizumab is independently selectedfrom at least 80%, at least 85%, at least 90%, at least 95% sequenceidentity, or at least 99% sequence identity. In certain aspects, theantibodies of the disclosure have VH and/or VL sequences having at least95%, at least 98% or at least 99% sequence identity to the VH and/or VLsequences of bevacizumab or ranibizumab.

In certain aspects, the antibodies of the disclosure have up to 17 aminoacid substitutions in their CDRs as compared to bevacizumab orranibizumab. Variant antibodies with 17 amino acid substitutions thatmaintain their target binding capability have been generated by Bostromet al., 2009, Science 323:1610-14.

In specific embodiments, an anti-VEGF antibody of the disclosure has,independently:

-   -   up to one, up to two, up to three, up to four, up to five, up to        six, up to seven, up to eight, up to nine or up to ten CDR-H1        substitutions as compared to the corresponding CDR of        bevacizumab or of ranibizumab;    -   up to one, up to two, up to three, up to four, up to five, up to        six, up to seven, up to eight, up to nine, up to ten, up to        eleven, up to twelve, up to thirteen, up to fourteen, up to        fifteen, up to sixteen or up to seventeen CDR-H2 substitutions        as compared to the corresponding CDR of bevacizumab or of        ranibizumab;    -   up to one, up to two, up to three, up to four, up to five, up to        six, up to seven, up to eight, up to nine, up to ten, up to        eleven, up to twelve, up to thirteen or up to fourteen CDR-H3        substitutions as compared to the corresponding CDR of        bevacizumab or of ranibizumab;    -   up to one, up to two, up to three, up to four, up to five, up to        six, up to seven, up to eight, up to nine, up to ten or up to        eleven CDR-L1 substitutions as compared to the corresponding CDR        of bevacizumab or of ranibizumab;    -   up to one, up to two, up to three, up to four, up to five, up to        six or up to seven CDR-L2 substitutions as compared to the        corresponding CDR of bevacizumab or of ranibizumab; and    -   up to one, up to two, up to three, up to four, up to five, up to        six, up to seven, up to eight or up to nine CDR-L3 substitutions        as compared to the corresponding CDR of bevacizumab or of        ranibizumab.

The present disclosure further provides pharmaceutical compositionscomprising modified anti-VEGF antibodies. In some aspects, thepharmaceutical compositions have increased affinity to VEGF and/orreduced immunogenicity as compared to bevacizumab or ranibizumab.

Nucleic acids comprising nucleotide sequences encoding the anti-VEGFantibodies of the disclosure are provided herein, as are vectorscomprising the nucleic acids. Additionally, prokaryotic and eukaryotichost cells transformed with a vector comprising a nucleotide sequenceencoding an anti-VEGF antibody are provided herein, as well aseukaryotic (such as mammalian) host cells engineered to express thenucleotide sequences. Methods of producing anti-VEGF antibodies byculturing host cells are also provided.

The anti-VEGF antibodies of the disclosure are useful in the treatmentof cancers (e.g., colon carcinoma, rectal carcinoma, non-small cell lungcancer, and breast cancer), retinal diseases (e.g., age-related maculardegeneration (“AMD”)), and immune disorders (e.g., rheumatoidarthritis).

In certain aspects, the anti-VEGF antibodies of the disclosure can beused in reduced dosages as compared to bevacizumab or ranibizumab, e.g.,at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, atleast 60%, at least 70%, at least 80% or at least 90% lower dosages.

It should be noted that the indefinite articles “a” and “an” and thedefinite article “the” are used in the present application, as is commonin patent applications, to mean one or more unless the context clearlydictates otherwise. Further, the term “or” is used in the presentapplication, as is common in patent applications, to mean thedisjunctive “or” or the conjunctive “and.”

All publications mentioned in this specification are herein incorporatedby reference. Any discussion of documents, acts, materials, devices,articles or the like that has been included in this specification issolely for the purpose of providing a context for the presentdisclosure. It is not to be taken as an admission that any or all ofthese matters form part of the prior art base or were common generalknowledge in the field relevant to the present disclosure as it existedanywhere before the priority date of this application.

The features and advantages of the disclosure will become furtherapparent from the following detailed description of embodiments thereof.

5. BRIEF DESCRIPTION OF THE TABLES AND FIGURES

FIGS. 1A-1D. FIG. 1A shows the amino acid sequences of the bevacizumabheavy and light chain variable regions, SEQ ID NO:1 and SEQ ID NO:2,respectively, with CDR regions in bold, underlined text. FIG. 1B showsthe CDR sequences and corresponding sequence identifiers of bevacizumab.FIG. 1C shows the amino acid sequences of the ranibizumab heavy andlight chains, SEQ ID NO:9 and SEQ ID NO:10, respectively, with CDRregions in bold, underlined text. FIG. 1D shows the CDR sequences andcorresponding sequence identifiers of ranibizumab.

FIGS. 2A-2B show bevacizumab VL peptide responses. FIG. 2A shows percentof donor responses to each VL peptide with a stimulation index of 2.95or greater. N=99 donors. FIG. 2B shows the average stimulation index forall 99 donors for each peptide plus or minus standard error.

FIGS. 3A-3B show bevacizumab VH peptide responses. FIG. 3A shows percentof donor responses to each VH peptide with a stimulation index of 2.95or greater. N=99 donors. FIG. 3B shows the average stimulation index forall 99 donors for each peptide plus or minus standard error.

FIGS. 4A-4C show CD4+ T cell responses to mutant bevacizumab epitopepeptides. Average responses to the unmodified parent epitope sequencesare indicated with open marks. Large circles indicate selected changesreferred to in FIG. 21. FIG. 4A is directed to VH CDR2 peptides; FIG. 4Bis directed to VH CDR3 peptides; and FIG. 4C is directed to VL CDR2peptides.

FIG. 5 shows the numbering of the amino acids in the heavy chain CDRs ofbevacizumab. CDRs 1-3 are disclosed as SEQ ID NOS:3-5, respectively.

FIG. 6 shows the numbering of the amino acids in the light chain CDRs ofbevacizumab. CDRs 1-3 are disclosed as SEQ ID NOS:6-8, respectively.

FIG. 7 shows bevacizumab VL peptides that were tested forimmunogenicity.

FIG. 8 shows bevacizumab VH peptides that were tested forimmunogenicity.

FIG. 9 shows identified CD4⁺ T cell epitope regions in bevacizumab. CDRregions are underlined.

FIG. 10 shows candidate mutations in CDR-H2 and CDR-H3 for loweringimmunogenicity of bevacizumab. The numbering of the amino acids in FIG.10 corresponds to Kabat numbering in the bevacizumab heavy chain.

FIG. 11 shows heavy chain CDR amino acid substitutions in bevacizumabresulting improved K_(D) as analyzed by surface plasmon resonace. Δk_(on) refers to fold improvement in k_(on) (mutant/WT). Δ k_(off)refers to fold improvement in k_(off)(WT/mutant). Δ K_(D) refers to theimprovement in the K_(D) in the mutant relative to wild type. Thenumbering of the amino acids in FIG. 11 corresponds to Kabat numberingin the bevacizumab heavy chain.

FIG. 12 shows mutations in the bevacizumab heavy chain CDRs thatpreliminary binding studies indicate increase the affinity towards VEGF(data not shown). The numbering of the amino acids in FIG. 12corresponds to Kabat numbering in the bevacizumab heavy chain.

FIG. 13 shows mutations in the bevacizumab heavy chain CDRs thatpreliminary studies indicate increase the affinity towards VEGF (datanot shown). The numbering of the amino acids in FIG. 13 corresponds toKabat numbering in the bevacizumab heavy chain.

FIG. 14 shows mutations in the bevacizumab heavy chain CDRs that do notimpact binding and can be incorporated into the antibodies of thedisclosure. The numbering of the amino acids in FIG. 14 corresponds toKabat numbering in the bevacizumab heavy chain.

FIG. 15 shows mutations in the bevacizumab light chain CDRs that do notimpact binding and can be incorporated into the antibodies of thedisclosure. The numbering of the amino acids in FIG. 15 corresponds toKabat numbering in the bevacizumab light chain.

FIG. 16A-16I show known mutations in bevacizumab heavy chain CDRs thatcan be incorporated into the antibodies of the disclosure. Each row inFIGS. 16A-16I includes a distinct known variant. For each variant, theknown CDR sequences are shaded. The sequence identifiers for eachvariant identified in FIGS. 16A-16I are set forth in FIGS. 24A-24I,respectively. The CDR-H1 is not shown. This partial sequence correspondsto SEQ ID NO:411. Although known mutations in CDR-H1 are shown in thecontext of this partial sequence, it is noted that the mutations existin the context of the full length CDR.

FIG. 17 shows known mutations in bevacizumab light chain CDRs that canbe incorporated into the antibodies of the disclosure. Each row in FIG.17 includes a distinct known variant. For each variant, the known CDRsequences are shaded. The sequence identifiers for each variantidentified in FIG. 17 is set forth in FIG. 24J.

FIG. 18 shows bevacizumab CDR2 VH peptides that were tested forimmunogenicity, wherein residues unchanged from SEQ ID NO:62 areindicated by a blank box. CD4+ T cell assay results are also provided.

FIG. 19 shows bevacizumab CDR3 VH peptides that were tested forimmunogenicity, wherein residues unchanged from SEQ ID NO:74 areindicated by a blank box. CD4+ T cell assay results are also provided.

FIG. 20 shows bevacizumab CDR2 VL peptides that were tested forimmunogenicity, wherein residues unchanged from SEQ ID NO:25 areindicated by a blank box. CD4+ T cell assay results are also provided.

FIG. 21 shows selected epitope modifications for the three CD4+ T cellepitopes in bevacizumab.

FIG. 22 shows single variable region mutants and their associated meanfluorescence intensity (MFI) score.

FIG. 23 shows combined variable region mutants and their associatedEC₅₀.

FIG. 24A-24J show the SEQ ID NOS, where known, corresponding to the CDRsof the bevacizumab variants listed in FIGS. 16A-16I and FIG. 17,respectively, N/A indicates an unknown CDR sequence.

6. DETAILED DESCRIPTION 6.1 Anti-VEGF Antibodies

The present disclosure provides anti-VEGF antibodies. Unless indicatedotherwise, the term “antibody” (Ab) refers to an immunoglobulin moleculethat specifically binds to, or is immunologically reactive with, aparticular antigen, and includes polyclonal, monoclonal, geneticallyengineered and otherwise modified forms of antibodies, including but notlimited to chimeric antibodies, humanized antibodies, heteroconjugateantibodies (e.g., bispecific antibodies, diabodies, triabodies, andtetrabodies), and antigen binding fragments of antibodies, includinge.g., Fab′, F(ab′)₂, Fab, Fv, rIgG, and scFv fragments. Moreover, unlessotherwise indicated, the term “monoclonal antibody” (mAb) is meant toinclude both intact molecules, as well as, antibody fragments (such as,for example, Fab and F(ab′)₂ fragments) which are capable ofspecifically binding to a protein. Fab and F(ab′)₂ fragments lack the Fcfragment of intact antibody, clear more rapidly from the circulation ofthe animal, and may have less non-specific tissue binding than an intactantibody (Wahl et al., 1983, J. Nucl. Med. 24:316).

The term “scFv” refers to a single chain Fv antibody in which thevariable domains of the heavy chain and the light chain from atraditional antibody have been joined to form one chain.

References to “VH” refer to the variable region of an immunoglobulinheavy chain of an antibody, including the heavy chain of an Fv, scFv, orFab. References to “VL” refer to the variable region of animmunoglobulin light chain, including the light chain of an Fv, scFv,dsFv or Fab. Antibodies (Abs) and immunoglobulins (Igs) areglycoproteins having the same structural characteristics. Whileantibodies exhibit binding specificity to a specific target,immunoglobulins include both antibodies and other antibody-likemolecules which lack target specificity. Native antibodies andimmunoglobulins are usually heterotetrameric glycoproteins of about150,000 Daltons, composed of two identical light (L) chains and twoidentical heavy (H) chains. Each heavy chain has at the amino terminus avariable domain (VH) followed by a number of constant domains. Eachlight chain has a variable domain at the amino terminus (VL) and aconstant domain at the carboxy terminus.

The anti-VEGF antibodies of the disclosure bind to human VEGF andinhibit VEGF receptor activity in a cell.

The anti-VEGF antibodies of the disclosure contain complementaritydetermining regions (CDRs) that are related in sequence to the CDRs ofthe antibody bevacizumab (also known as Avastin®) and/or ranibizumab(also known as Lucentis®).

CDRs are also known as hypervariable regions both in the light chain andthe heavy chain variable domains. The more highly conserved portions ofvariable domains are called the framework (FR). As is known in the art,the amino acid position/boundary delineating a hypervariable region ofan antibody can vary, depending on the context and the variousdefinitions known in the art. Some positions within a variable domainmay be viewed as hybrid hypervariable positions in that these positionscan be deemed to be within a hypervariable region under one set ofcriteria while being deemed to be outside a hypervariable region under adifferent set of criteria. One or more of these positions can also befound in extended hypervariable regions. The disclosure providesantibodies comprising modifications in these hybrid hypervariablepositions. The variable domains of native heavy and light chains eachcomprise four FR regions, largely by adopting a β-sheet configuration,connected by three CDRs, which form loops connecting, and in some casesforming part of, the β-sheet structure. The CDRs in each chain are heldtogether in close proximity by the FR regions in the orderFR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 and, with the CDRs from the other chain,contribute to the formation of the target binding site of antibodies(see Kabat et al., Sequences of Proteins of Immunological Interest(National Institute of Health, Bethesda, Md. 1987). As used herein,numbering of immunoglobulin amino acid residues is done according to theimmunoglobulin amino acid residue numbering system of Kabat et al.,unless otherwise indicated.

The sequences of the heavy and light chain variable regions ofbevacizumab are represented by SEQ ID NO:1 and SEQ ID NO:2,respectively. The sequences of the heavy and light chain variableregions are also depicted in FIG. 1A. The sequences of the CDRs ofbevacizumab, and their corresponding identifiers, are presented in FIG.1B. Any nucleotide sequences encoding SEQ ID NO:1 or SEQ ID NO:2 can beused in the compositions and methods of the present disclosure.

The sequences of the heavy and light chains of ranibizumab arerepresented by SEQ ID NO:9 and SEQ ID NO:10, respectively. The sequencesof the heavy and light chains are also depicted in FIG. 1C. Thesequences of the CDRs of ranibizumab, and their correspondingidentifiers, are presented in FIG. 1D. Any nucleotide sequences encodingSEQ ID NO:9 or SEQ ID NO:10 can be used in the compositions and methodsof the present disclosure.

The present disclosure further provides anti-VEGF antibody fragmentscomprising CDR sequences that are related to the CDR sequences ofbevacizumab and ranibizumab. The term “antibody fragment” refers to aportion of a full-length antibody, generally the target binding orvariable region. Examples of antibody fragments include Fab, Fab′,F(ab′)₂ and Fv fragments. An “Fv” fragment is the minimum antibodyfragment which contains a complete target recognition and binding site.This region consists of a dimer of one heavy and one light chainvariable domain in a tight, non-covalent association (VH-VL dimer). Itis in this configuration that the three CDRs of each variable domaininteract to define a target binding site on the surface of the VH-VLdimer. Often, the six CDRs confer target binding specificity to theantibody. However, in some instances even a single variable domain (orhalf of an Fv comprising only three CDRs specific for a target) can havethe ability to recognize and bind target. “Single-chain Fv” or “scFv”antibody fragments comprise the VH and VL domains of an antibody in asingle polypeptide chain. Generally, the Fv polypeptide furthercomprises a polypeptide linker between the VH and VL domain whichenables the scFv to form the desired structure for target binding.“Single domain antibodies” are composed of a single VH or VL domainswhich exhibit sufficient affinity to the target. In a specificembodiment, the single domain antibody is a camelid antibody (see, e.g.,Riechmann, 1999, Journal of Immunological Methods 231:25-38).

The Fab fragment contains the constant domain of the light chain and thefirst constant domain (CH₁) of the heavy chain. Fab′ fragments differfrom Fab fragments by the addition of a few residues at the carboxylterminus of the heavy chain CH₁ domain including one or more cysteinesfrom the antibody hinge region. F(ab′) fragments are produced bycleavage of the disulfide bond at the hinge cysteines of the F(ab′)₂pepsin digestion product. Additional chemical couplings of antibodyfragments are known to those of ordinary skill in the art.

In certain embodiments, the anti-VEGF antibodies of the disclosure aremonoclonal antibodies. The term “monoclonal antibody” as used herein isnot limited to antibodies produced through hybridoma technology. Theterm “monoclonal antibody” refers to an antibody that is derived from asingle clone, including any eukaryotic, prokaryotic, or phage clone, andnot the method by which it is produced. Monoclonal antibodies useful inconnection with the present disclosure can be prepared using a widevariety of techniques known in the art including the use of hybridoma,recombinant, and phage display technologies, or a combination thereof.The anti-VEGF antibodies of the disclosure include chimeric, primatized,humanized, or human antibodies.

The anti-VEGF antibodies of the disclosure can be chimeric antibodies.The term “chimeric” antibody as used herein refers to an antibody havingvariable sequences derived from a non-human immunoglobulin, such as rator mouse antibody, and human immunoglobulin constant regions, typicallychosen from a human immunoglobulin template. Methods for producingchimeric antibodies are known in the art. See, e.g., Morrison, 1985,Science 229(4719):1202-7; Oi et al., 1986, BioTechniques 4:214-221;Gillies et al., 1985, J. Immunol. Methods 125:191-202; U.S. Pat. Nos.5,807,715; 4,816,567; and 4,816397, which are incorporated herein byreference in their entireties.

The anti-VEGF antibodies of the disclosure can be humanized. “Humanized”forms of non-human (e.g., murine) antibodies are chimericimmunoglobulins, immunoglobulin chains or fragments thereof (such as Fv,Fab, Fab′, F(ab′)₂ or other target-binding subdomains of antibodies)which contain minimal sequences derived from non-human immunoglobulin.In general, the humanized antibody will comprise substantially all of atleast one, and typically two, variable domains, in which all orsubstantially all of the CDR regions correspond to those of a non-humanimmunoglobulin and all or substantially all of the FR regions are thoseof a human immunoglobulin sequence. The humanized antibody can alsocomprise at least a portion of an immunoglobulin constant region (Fc),typically that of a human immunoglobulin consensus sequence. Methods ofantibody humanization are known in the art. See, e.g., Riechmann et al.,1988, Nature 332:323-7; U.S. Pat. Nos. 5,530,101; 5,585,089; 5,693,761;5,693,762; and 6,180,370 to Queen et al.; EP239400; PCT publication WO91/09967; U.S. Pat. No. 5,225,539; EP592106; EP519596; Padlan, 1991,Mol. Immunol., 28:489-498; Studnicka et al., 1994, Prot. Eng. 7:805-814;Roguska et al., 1994, Proc. Natl. Acad. Sci. 91:969-973; and U.S. Pat.No. 5,565,332, all of which are hereby incorporated by reference intheir entireties.

The anti-VEGF antibodies of the disclosure can be human antibodies.Completely “human” anti-VEGF antibodies can be desirable for therapeutictreatment of human patients. As used herein, “human antibodies” includeantibodies having the amino acid sequence of a human immunoglobulin andinclude antibodies isolated from human immunoglobulin libraries or fromanimals transgenic for one or more human immunoglobulin and that do notexpress endogenous immunoglobulins. Human antibodies can be made by avariety of methods known in the art including phage display methodsusing antibody libraries derived from human immunoglobulin sequences.See U.S. Pat. Nos. 4,444,887 and 4,716,111; and PCT publications WO98/46645; WO 98/50433; WO 98/24893; WO 98/16654; WO 96/34096; WO96/33735; and WO 91/10741, each of which is incorporated herein byreference in its entirety. Human antibodies can also be produced usingtransgenic mice which are incapable of expressing functional endogenousimmunoglobulins, but which can express human immunoglobulin genes. See,e.g., PCT publications WO 98/24893; WO 92/01047; WO 96/34096; WO96/33735; U.S. Pat. Nos. 5,413,923; 5,625,126; 5,633,425; 5,569,825;5,661,016; 5,545,806; 5,814,318; 5,885,793; 5,916,771; and 5,939,598,which are incorporated by reference herein in their entireties. Inaddition, companies such as Medarex (Princeton, N.J.), Astellas Pharma(Deerfield, Ill.), Amgen (Thousand Oaks, Calif.) and Regeneron(Tarrytown, N.Y.) can be engaged to provide human antibodies directedagainst a selected antigen using technology similar to that describedabove. Completely human antibodies that recognize a selected epitope canbe generated using a technique referred to as “guided selection.” Inthis approach a selected non-human monoclonal antibody, e.g., a mouseantibody, is used to guide the selection of a completely human antibodyrecognizing the same epitope (Jespers et al., 1988, Biotechnology12:899-903).

The anti-VEGF antibodies of the disclosure can be primatized. The term“primatized antibody” refers to an antibody comprising monkey variableregions and human constant regions. Methods for producing primatizedantibodies are known in the art. See e.g., U.S. Pat. Nos. 5,658,570;5,681,722; and 5,693,780, which are incorporated herein by reference intheir entireties.

The anti-VEGF antibodies of the disclosure can be bispecific antibodies.Bispecific antibodies are monoclonal, often human or humanized,antibodies that have binding specificities for at least two differentantigens. In the present disclosure, one of the binding specificitiescan be directed towards VEGF, the other can be for any other antigen,e.g., for a cell-surface protein, receptor, receptor subunit,tissue-specific antigen, virally derived protein, virally encodedenvelope protein, bacterially derived protein, or bacterial surfaceprotein, etc. In a specific embodiment, an antibody of the disclosure isa bispecific antibody with binding specificites for both VEGF and CD3.

The anti-VEGF antibodies of the disclosure include derivatizedantibodies. For example, but not by way of limitation, derivatizedantibodies are typically modified by glycosylation, acetylation,pegylation, phosphorylation, amidation, derivatization by knownprotecting/blocking groups, proteolytic cleavage, linkage to a cellularligand or other protein (see Section 6.6 for a discussion of antibodyconjugates), etc. Any of numerous chemical modifications can be carriedout by known techniques, including, but not limited to, specificchemical cleavage, acetylation, formylation, metabolic synthesis oftunicamycin, etc. Additionally, the derivative can contain one or morenon-natural amino acids, e.g., using ambrx technology (see, e.g.,Wolfson, 2006, Chem. Biol. 13(10):1011-2).

In yet another embodiment of the disclosure, the anti-VEGF antibodies orfragments thereof can be antibodies or antibody fragments whose sequencehas been modified to alter at least one constant region-mediatedbiological effector function relative to the corresponding wild typesequence.

For example, in some embodiments, an anti-VEGF antibody of thedisclosure can be modified to reduce at least one constantregion-mediated biological effector function relative to an unmodifiedantibody, e.g., reduced binding to the Fc receptor (FcγR). FcγR bindingcan be reduced by mutating the immunoglobulin constant region segment ofthe antibody at particular regions necessary for FcγR interactions (seee.g., Canfield and Morrison, 1991, J. Exp. Med. 173:1483-1491; and Lundet al., 1991, J. Immunol. 147:2657-2662). Reduction in FcγR bindingability of the antibody can also reduce other effector functions whichrely on FcγR interactions, such as opsonization, phagocytosis andantigen-dependent cellular cytotoxicity (“ADCC”).

In other embodiments, an anti-VEGF antibody of the disclosure can bemodified to acquire or improve at least one constant region-mediatedbiological effector function relative to an unmodified antibody, e.g.,to enhance FcγR interactions (see, e.g., US 2006/0134709). For example,an anti-VEGF antibody of the disclosure can have a constant region thatbinds FcγRIIA, FcγRIIB and/or FcγRIIIA with greater affinity than thecorresponding wild type constant region.

Thus, antibodies of the disclosure can have alterations in biologicalactivity that result in increased or decreased opsonization,phagocytosis, or ADCC. Such alterations are known in the art. Forexample, modifications in antibodies that reduce ADCC activity aredescribed in U.S. Pat. No. 5,834,597. An exemplary ADCC lowering variantcorresponds to “mutant 3” shown in FIG. 4 of U.S. Pat. No. 5,834,597, inwhich residue 236 is deleted and residues 234, 235 and 237 (using EUnumbering) are substituted with alanines.

In some embodiments, the anti-VEGF antibodies of the disclosure have lowlevels of or lack fucose. Antibodies lacking fucose have been correlatedwith enhanced ADCC (activity, especially at low doses of antibody. SeeShields et al., 2002, J. Biol. Chem. 277:26733-26740; Shinkawa et al.,2003, J. Biol. Chem. 278:3466-73. Methods of preparing fucose-lessantibodies include growth in rat myeloma YB2/0 cells (ATCC CRL 1662).YB2/0 cells express low levels of FUT8 mRNA, which encodesα-1,6-fucosyltransferase, an enzyme necessary for fucosylation ofpolypeptides.

In yet another aspect, the anti-VEGF antibodies or fragments thereof canbe antibodies or antibody fragments that have been modified to increaseor reduce their binding affinities to the fetal Fc receptor, FcRn, forexample by mutating the immunoglobulin constant region segment atparticular regions involved in FcRn interactions (see, e.g., WO2005/123780). In particular embodiments, an anti-VEGF antibody of theIgG class is mutated such that at least one of amino acid residues 250,314, and 428 of the heavy chain constant region is substituted alone, orin any combinations thereof, such as at positions 250 and 428, or atpositions 250 and 314, or at positions 314 and 428, or at positions 250,314, and 428, with positions 250 and 428 a specific combination. Forposition 250, the substituting amino acid residue can be any amino acidresidue other than threonine, including, but not limited to, alanine,cysteine, aspartic acid, glutamic acid, phenylalanine, glycine,histidine, isoleucine, lysine, leucine, methionine, asparagine, proline,glutamine, arginine, serine, valine, tryptophan, or tyrosine. Forposition 314, the substituting amino acid residue can be any amino acidresidue other than leucine, including, but not limited to, alanine,cysteine, aspartic acid, glutamic acid, phenylalanine, glycine,histidine, isoleucine, lysine, methionine, asparagine, proline,glutamine, arginine, serine, threonine, valine, tryptophan, or tyrosine.For position 428, the substituting amino acid residues can be any aminoacid residue other than methionine, including, but not limited to,alanine, cysteine, aspartic acid, glutamic acid, phenylalanine, glycine,histidine, isoleucine, lysine, leucine, asparagine, proline, glutamine,arginine, serine, threonine, valine, tryptophan, or tyrosine. Specificcombinations of suitable amino acid substitutions are identified inTable 1 of U.S. Pat. No. 7,217,797, which table is incorporated byreference herein in its entirety. Such mutations increase the antibody'sbinding to FcRn, which protects the antibody from degradation andincreases its half-life.

In yet other aspects, an anti-VEGF antibody has one or more amino acidsinserted into one or more of its hypervariable regions, for example asdescribed in Jung and Pliickthun, 1997, Protein Engineering10(9):959-966; Yazaki et al., 2004, Protein Eng Des. Sel. 17(5):481-9.Epub 2004 Aug. 17; and US 2007/0280931.

In various embodiments, the anti-VEGF antibodies or fragments thereofcan be antibodies or antibody fragments that have been modified forincreased expression in heterologous hosts. In certain embodiments, theanti-VEGF antibodies or fragments thereof can be antibodies or antibodyfragments that have been modified for increased expression in and/orsecretion from heterologous host cells. In some embodiments, theanti-VEGF antibodies or fragments thereof are modified for increasedexpression in bacteria, such as E. coli. In other embodiments, theanti-VEGF antibodies or fragments thereof are modified for increasedexpression in yeast (Kieke et al., 1999, Proc. Nat'l Acad. Sci. USA96:5651-5656). In still other embodiments, the anti-VEGF antibodies orfragments thereof are modified for increased expression in insect cells.In additional embodiments, the anti-VEGF antibodies or fragments thereofare modified for increased expression in mammalian cells, such as CHOcells.

In certain embodiments, the anti-VEGF antibodies or fragments thereofcan be antibodies or antibody fragments that have been modified toincrease stability of the antibodies during production. In someembodiments, the antibodies or fragments thereof can be modified toreplace one or more amino acids such as asparagine or glutamine that aresusceptible to nonenzymatic deamidation with amino acids that do notundergo deamidation (Huang et al., 2005, Anal. Chem. 77:1432-1439). Inother embodiments, the antibodies or fragments thereof can be modifiedto replace one or more amino acids that is susceptible to oxidation,such as methionine, cysteine or tryptophan, with an amino acid that doesnot readily undergo oxidation. In still other embodiments, theantibodies or fragments thereof can be modified to replace one or moreamino acids that is susceptible to cyclization, such as asparagine orglutamic acid, with an amino acid that does not readily undergocyclization.

6.2 Nucleic Acids and Expression Systems

The present disclosure encompasses nucleic acid molecules and host cellsencoding the anti-VEGF antibodies of the disclosure.

An anti-VEGF antibody of the disclosure can be prepared by recombinantexpression of immunoglobulin light and heavy chain genes in a host cell.To express an antibody recombinantly, a host cell is transfected withone or more recombinant expression vectors carrying DNA fragmentsencoding the immunoglobulin light and heavy chains of the antibody suchthat the light and heavy chains are expressed in the host cell and,optionally, secreted into the medium in which the host cells arecultured, from which medium the antibodies can be recovered. Standardrecombinant DNA methodologies are used to obtain antibody heavy andlight chain genes, incorporate these genes into recombinant expressionvectors and introduce the vectors into host cells, such as thosedescribed in Molecular Cloning; A Laboratory Manual, Second Edition(Sambrook, Fritsch and Maniatis (eds), Cold Spring Harbor, N.Y., 1989),Current Protocols in Molecular Biology (Ausubel, F. M. et al., eds.,Greene Publishing Associates, 1989) and in U.S. Pat. No. 4,816,397.

In one embodiment, the anti-VEGF antibodies are similar to bevacizumabor ranibizumab but for changes in one or more CDRs. In anotherembodiment, the anti-VEGF antibodies are similar to bevacizumab orranibizumab but for changes in one or more framework regions. In yetanother embodiment, the anti-VEGF antibodies are similar to bevacizumabor ranibizumab but for changes in one or more CDRs and in one or moreframework regions. Such antibodies are referred to herein collectivelyas having “bevacizumab-related” or “ranibizumab-related” sequences andare sometimes referenced simply as anti-VEGF antibodies of thedisclosure. To generate nucleic acids encoding anti-VEGF antibodies, DNAfragments encoding the light and heavy chain variable regions are firstobtained. These DNAs can be obtained by amplification and modificationof germline DNA or cDNA encoding light and heavy chain variablesequences, for example using the polymerase chain reaction (PCR).Germline DNA sequences for human heavy and light chain variable regiongenes are known in the art (see, e.g., the “VBASE” human germlinesequence database; see also Kabat, E. A. et al., 1991, Sequences ofProteins of Immunological Interest, Fifth Edition, U.S. Department ofHealth and Human Services, NIH Publication No. 91-3242; Tomlinson etal., 1992, J. Mol. Biol. 22T:116-198; and Cox et al., 1994, Eur. J.Immunol. 24:827-836; the contents of each of which are incorporatedherein by reference). A DNA fragment encoding the heavy or light chainvariable region of bevacizumab or ranibizumab can be synthesized andused as a template for mutagenesis to generate a variant as describedherein using routine mutagenesis techniques; alternatively, a DNAfragment encoding the variant can be directly synthesized.

Once DNA fragments encoding anti-VEGF VH and VL segments are obtained,these DNA fragments can be further manipulated by standard recombinantDNA techniques, for example to convert the variable region genes tofull-length antibody chain genes, to Fab fragment genes or to a scFvgene. In these manipulations, a VL- or VH-encoding DNA fragment isoperatively linked to another DNA fragment encoding another protein,such as an antibody constant region or a flexible linker. The term“operatively linked,” as used in this context, is intended to mean thatthe two DNA fragments are joined such that the amino acid sequencesencoded by the two DNA fragments remain in-frame.

The isolated DNA encoding the VH region can be converted to afull-length heavy chain gene by operatively linking the VH-encoding DNAto another DNA molecule encoding heavy chain constant regions (CH₁, CH₂,CH₃ and, optionally, CH₄). The sequences of human heavy chain constantregion genes are known in the art (see, e.g., Kabat, E. A. et al., 1991,Sequences of Proteins of Immunological Interest, Fifth Edition, U.S.Department of Health and Human Services, NIH Publication No. 91-3242)and DNA fragments encompassing these regions can be obtained by standardPCR amplification. The heavy chain constant region can be an IgG₁, IgG₂,IgG₃, IgG₄, IgA, IgE, IgM or IgD constant region, but in certainembodiments is an IgG₁ or IgG₄ constant region. For a Fab fragment heavychain gene, the VH-encoding DNA can be operatively linked to another DNAmolecule encoding only the heavy chain CH₁ constant region.

The isolated DNA encoding the VL region can be converted to afull-length light chain gene (as well as a Fab light chain gene) byoperatively linking the VL-encoding DNA to another DNA molecule encodingthe light chain constant region, CL. The sequences of human light chainconstant region genes are known in the art (see, e.g., Kabat, E. A. etal., 1991, Sequences of Proteins of Immunological Interest, FifthEdition (U.S. Department of Health and Human Services, NIH PublicationNo. 91-3242)) and DNA fragments encompassing these regions can beobtained by standard PCR amplification. The light chain constant regioncan be a kappa or lambda constant region, but in certain embodiments isa kappa constant region. To create a scFv gene, the VH- and VL-encodingDNA fragments are operatively linked to another fragment encoding aflexible linker, e.g., encoding the amino acid sequence (Gly₄˜Ser)₃,such that the VH and VL sequences can be expressed as a contiguoussingle-chain protein, with the VL and VH regions joined by the flexiblelinker (see, e.g., Bird et al., 1988, Science 242:423-426; Huston etal., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; McCafferty et al.,1990, Nature 348:552-554).

To express the anti-VEGF antibodies of the disclosure, DNAs encodingpartial or full-length light and heavy chains, obtained as describedabove, are inserted into expression vectors such that the genes areoperatively linked to transcriptional and translational controlsequences. In this context, the term “operatively linked” is intended tomean that an antibody gene is ligated into a vector such thattranscriptional and translational control sequences within the vectorserve their intended function of regulating the transcription andtranslation of the antibody gene. The expression vector and expressioncontrol sequences are chosen to be compatible with the expression hostcell used. The antibody light chain gene and the antibody heavy chaingene can be inserted into separate vectors or, more typically, bothgenes are inserted into the same expression vector.

The antibody genes are inserted into the expression vector by standardmethods (e.g., ligation of complementary restriction sites on theantibody gene fragment and vector, or blunt end ligation if norestriction sites are present). Prior to insertion of the light or heavychain sequences of the anti-VEGF antibodies of the disclosure, theexpression vector can already carry antibody constant region sequences.For example, one approach to converting the anti-VEGF VH and VLsequences to full-length antibody genes is to insert them intoexpression vectors already encoding heavy chain constant and light chainconstant regions, respectively, such that the VH segment is operativelylinked to the CH segment(s) within the vector and the VL segment isoperatively linked to the CL segment within the vector. Additionally oralternatively, the recombinant expression vector can encode a signalpeptide that facilitates secretion of the antibody chain from a hostcell. The antibody chain gene can be cloned into the vector such thatthe signal peptide is linked in-frame to the amino terminus of theantibody chain gene. The signal peptide can be an immunoglobulin signalpeptide or a heterologous signal peptide (i.e., a signal peptide from anon-immunoglobulin protein).

In addition to the antibody chain genes, the recombinant expressionvectors of the disclosure carry regulatory sequences that control theexpression of the antibody chain genes in a host cell. The term“regulatory sequence” is intended to include promoters, enhancers andother expression control elements (e.g., polyadenylation signals) thatcontrol the transcription or translation of the antibody chain genes.Such regulatory sequences are described, for example, in Goeddel, GeneExpression Technology: Methods in Enzymology 185 (Academic Press, SanDiego, Calif., 1990). It will be appreciated by those skilled in the artthat the design of the expression vector, including the selection ofregulatory sequences may depend on such factors as the choice of thehost cell to be transformed, the level of expression of protein desired,etc. Suitable regulatory sequences for mammalian host cell expressioninclude viral elements that direct high levels of protein expression inmammalian cells, such as promoters and/or enhancers derived fromcytomegalovirus (CMV) (such as the CMV promoter/enhancer), Simian Virus40 (SV40) (such as the SV40 promoter/enhancer), adenovirus, (e.g., theadenovirus major late promoter (AdMLP)) and polyoma. For furtherdescription of viral regulatory elements, and sequences thereof, seee.g., U.S. Pat. No. 5,168,062 by Stinski, U.S. Pat. No. 4,510,245 byBell et al., and U.S. Pat. No. 4,968,615 by Schaffner et al.,

In addition to the antibody chain genes and regulatory sequences, therecombinant expression vectors of the disclosure can carry additionalsequences, such as sequences that regulate replication of the vector inhost cells (e.g., origins of replication) and selectable marker genes.The selectable marker gene facilitates selection of host cells intowhich the vector has been introduced (see e.g., U.S. Pat. Nos.4,399,216, 4,634,665 and 5,179,017, all by Axel et al.). For example,typically the selectable marker gene confers resistance to drugs, suchas G418, puromycin, blasticidin, hygromycin or methotrexate, on a hostcell into which the vector has been introduced. Suitable selectablemarker genes include the dihydrofolate reductase (DHFR) gene (for use inDHFR⁻ host cells with methotrexate selection/amplification) and the neogene (for G418 selection). For expression of the light and heavy chains,the expression vector(s) encoding the heavy and light chains istransfected into a host cell by standard techniques. The various formsof the term “transfection” are intended to encompass a wide variety oftechniques commonly used for the introduction of exogenous DNA into aprokaryotic or eukaryotic host cell, e.g., electroporation, lipofection,calcium-phosphate precipitation, DEAE-dextran transfection and the like.

It is possible to express the antibodies of the disclosure in eitherprokaryotic or eukaryotic host cells. In certain embodiments, expressionof antibodies is performed in eukaryotic cells, e.g., mammalian hostcells, for optimal secretion of a properly folded and immunologicallyactive antibody. Exemplary mammalian host cells for expressing therecombinant antibodies of the disclosure include Chinese Hamster Ovary(CHO cells) (including DHFR⁻ CHO cells, described in Urlaub and Chasin,1980, Proc. Natl. Acad. Sci. USA 77:4216-4220, used with a DHFRselectable marker, e.g., as described in Kaufman and Sharp, 1982, Mol.Biol. 159:601-621), NS0 myeloma cells, COS cells, 293 cells and SP2/0cells. When recombinant expression vectors encoding antibody genes areintroduced into mammalian host cells, the antibodies are produced byculturing the host cells for a period of time sufficient to allow forexpression of the antibody in the host cells or secretion of theantibody into the culture medium in which the host cells are grown.Antibodies can be recovered from the culture medium using standardprotein purification methods. Host cells can also be used to produceportions of intact antibodies, such as Fab fragments or scFv molecules.It is understood that variations on the above procedure are within thescope of the present disclosure. For example, it can be desirable totransfect a host cell with DNA encoding either the light chain or theheavy chain (but not both) of an anti-VEGF antibody of this disclosure.

Recombinant DNA technology can also be used to remove some or all of theDNA encoding either or both of the light and heavy chains that is notnecessary for binding to VEGF. The molecules expressed from suchtruncated DNA molecules are also encompassed by the antibodies of thedisclosure.

In addition, bifunctional antibodies can be produced in which one heavyand one light chain are an antibody of the disclosure and the otherheavy and light chain are specific for an antigen other than VEGF bycrosslinking an antibody of the disclosure to a second antibody bystandard chemical crosslinking methods. Bifunctional antibodies can alsobe made by expressing a nucleic acid engineered to encode a bifunctionalantibody.

In certain embodiments, dual specific antibodies, i.e., antibodies thatbind VEGF and an unrelated antigen using the same binding site, can beproduced by mutating amino acid residues in the light chain and/or heavychain CDRs. In various embodiments, dual specific antibodies that bindtwo antigens, such as HER2 and VEGF, can be produced by mutating aminoacid residues in the periphery of the antigen binding site (Bostrom etal., 2009, Science 323:1610-1614). Dual functional antibodies can bemade by expressing a nucleic acid engineered to encode a dual specificantibody.

For recombinant expression of an anti-VEGF antibody of the disclosure,the host cell can be co-transfected with two expression vectors of thedisclosure, the first vector encoding a heavy chain derived polypeptideand the second vector encoding a light chain derived polypeptide.Typically, the two vectors each contain a separate selectable marker.Alternatively, a single vector can be used which encodes both heavy andlight chain polypeptides.

Once a nucleic acid encoding one or more portions of an anti-VEGFantibody is generated, further alterations or mutations can beintroduced into the coding sequence, for example to generate nucleicacids encoding antibodies with different CDR sequences, antibodies withreduced affinity to the Fc receptor, or antibodies of differentsubclasses.

The anti-VEGF antibodies of the disclosure can also be produced bychemical synthesis (e.g., by the methods described in Solid PhasePeptide Synthesis, 2^(nd) ed., 1984 The Pierce Chemical Co., Rockford,Ill.). Variant antibodies can also be generated using a cell-freeplatform (see, e.g., Chu et al., 2001, Biochemia No. 2 (Roche MolecularBiologicals)).

Once an anti-VEGF antibody of the disclosure has been produced byrecombinant expression, it can be purified by any method known in theart for purification of an immunoglobulin molecule, for example, bychromatography (e.g., ion exchange, affinity, particularly by affinityfor VEGF after Protein A or Protein G selection, and sizing columnchromatography), centrifugation, differential solubility, or by anyother standard technique for the purification of proteins. Further, theanti-VEGF antibodies of the present disclosure or fragments thereof canbe fused to heterologous polypeptide sequences described herein orotherwise known in the art to facilitate purification.

Once isolated, an anti-VEGF antibody can, if desired, be furtherpurified, e.g., by high performance liquid chromatography (See, e.g.,Fisher, Laboratory Techniques In Biochemistry And Molecular Biology(Work and Burdon, eds., Elsevier, 1980), or by gel filtrationchromatography on a Superdex™ 75 column (Pharmacia Biotech AB, Uppsala,Sweden).

6.3 Biological Activities of Anti-VEGF Antibodies

In certain embodiments, the anti-VEGF antibodies of the disclosure havecertain biological activities, such as competing with bevacizumab orranibizumab for binding to VEGF or neutralizing VEGF activity.

Accordingly, in certain embodiments, anti-VEGF antibodies of thedisclosure compete with bevacizumab or ranibizumab for binding to VEGF.The ability to compete for binding to VEGF can be tested using acompetition assay. In one example of a competition assay, VEGF isadhered onto a solid surface, e.g., a microwell plate, by contacting theplate with a solution of VEGF (e.g., at a concentration of 1 μg/mL inPBS over night at 4° C.). The plate is washed (e.g., 0.1% Tween 20 inPBS) and blocked (e.g., in Superblock, Thermo Scientific, Rockford,Ill.). A mixture of sub-saturating amount of biotinylated bevacizumab(80 ng/mL) or an equivalent amount of biotinylated ranibizumab andunlabeled bevacizumab (or ranibizumab as the case may be) (the“reference” antibody) or competing anti-VEGF antibody (the “test”antibody) antibody in serial dilution (e.g., at a concentration of 2.8μg/mL, 8.3 μg/mL, or 25 μg/mL) in ELISA buffer (e.g., 1% BSA and 0.1%Tween 20 in PBS) is added to wells and plates are incubated for 1 hourwith gentle shaking. The plate is washed, 1 μg/mL HRP-conjugatedStreptavidin diluted in ELISA buffer is added to each well and theplates incubated for 1 hour. Plates are washed and bound antibodies weredetected by addition of substrate (e.g., TMB, Biofx Laboratories Inc.,Owings Mills, Md.). The reaction is terminated by addition of stopbuffer (e.g., Bio FX Stop Reagents, Biofx Laboratories Inc., OwingsMills, Md.) and the absorbance is measured at 650 nm using microplatereader (e.g., VERSAmax, Molecular Devices, Sunnyvale, Calif.).Variations on this competition assay can also be used to testcompetition between an anti-VEGF antibody of the disclosure andbevacizumab or ranibizumab. For example, in certain aspects, theanti-VEGF antibody is used as a reference antibody and bevacizumab orranibizumab is used as a test antibody. Additionally, instead of solubleVEGF, membrane-bound VEGF expressed on the surfaces of cell (for examplemammalian cells) in culture can be used. Other formats for competitionassays are known in the art and can be employed.

In various embodiments, an anti-VEGF antibody of the disclosure reducesthe binding of labeled bevacizumab or ranibizumab by at least 30%, by atleast 40%, by at least 50%, by at least 60%, by at least 70%, by atleast 80%, by at least 90%, by at least 95%, by at least 99% or by apercentage ranging between any of the foregoing values (e.g., ananti-VEGF antibody of the disclosure reduces the binding of labeledbevacizumab or ranibizumab by 50% to 70%) when the anti-VEGF antibody isused at a concentration of 0.08 μg/mL, 0.4 μg/mL, 2 μg/mL, 10 μg/mL, 50μg/mL, 100 μg/mL or at a concentration ranging between any of theforegoing values (e.g., at a concentration ranging from 2 μg/mL to 10μg/mL).

In other embodiments, bevacizumab or ranibizumab reduces the binding ofa labeled anti-VEGF antibody of the disclosure by at least 40%, by atleast 50%, by at least 60%, by at least 70%, by at least 80%, by atleast 90%, or by a percentage ranging between any of the foregoingvalues (e.g., bevacizumab or ranibizumab reduces the binding of alabeled an anti-VEGF antibody of the disclosure by 50% to 70%) whenbevacizumab or ranibizumab is used at a concentration of 0.4 μg/mL, 2μg/mL, 10 μg/mL, 50 μg/mL, 250 μg/mL or at a concentration rangingbetween any of the foregoing values (e.g., at a concentration rangingfrom 2 μg/mL to 10 μg/mL).

In other aspects, an anti-VEGF antibody of the disclosure inhibits (orneutralizes) VEGF activity in a range of in vitro assays, such as cellproliferation or cell migration. For example, in one embodiment, theVEGF activity assayed is induction of endothelial cell (“EC”)proliferation (see, e.g., protocol of Qin et al., 2006, J. Biol. Chem.281:32550-32558). In another embodiment, the VEGF activity assayed isinduction of EC migration (see, e.g., the in vitro scratch assayprotocol described of Liang et al., 2007, Nat. Protoc. 2:329-333). In aspecific embodiment, an anti-VEGF antibody is tested for the ability toreverse proliferation and cell migration stimulated by VEGF anddelocalization of tight junction proteins induced by VEGF₁₆₅ inimmortalized bovine retinal endothelial cells (Deissler et al., 2008,British Journal of Opthalmology 92:839-843). In yet another embodiment,the neutralization of VEGF activity is assayed using a reporter assay(see, e.g., Yohno et al., 2003, Biological & Pharmaceutical Bulletin26(4):417-20 and U.S. Pat. No. 6,787,323).

Other formats for VEGF neutralization assays are known in the art andcan be employed.

In various embodiments, an anti-VEGF antibody of the disclosureneutralizes VEGF by at least 30%, by at least 40%, by at least 50%, byat least 60%, by at least 70%, by at least 80%, by at least 90%, or by apercentage ranging between any of the foregoing values (e.g., ananti-VEGF antibody of the disclosure neutralizes VEGF activity by 50% to70%) when the anti-VEGF antibody is used at a concentration of 2 ng/mL,5 ng/mL, 10 ng/mL, 20 ng/mL, 0.1 μg/mL, 0.2 μg/mL, 1 μg/mL, 2 μg/mL, 5μg/mL, 10 μg/mL, 20 μg/mL, or at a concentration ranging between any ofthe foregoing values (e.g., at a concentration ranging from 1 μg/mL to 5μg/mL).

In some embodiments, an anti-VEGF antibody of the disclosure is at least0.7-fold as effective, 0.8-fold as effective, at least 0.9-fold aseffective, at least 1-fold as effective, at least 1.1-fold as effective,at least 1.25-fold as effective, at least 1.5-fold as effective, atleast 2-fold as effective, at least 5-fold as effective, at least10-fold as effective, at least 20-fold as effective, at least 50-fold aseffective, at least 100-fold as effective, at least 200-fold aseffective, at least 500-fold as effective, at least 1000-fold aseffective as bevacizumab or ranibizumab at neutralizing VEGF, or havingan effectiveness at neutralizing VEGF relative to bevacizumab orranibizumab ranging between any pair of the foregoing values (e.g.,0.9-fold to 5-fold as effective as bevacizumab or ranibizumab or 2-foldto 50-fold as effective as bevacizumab or ranibizumab in neutralizingVEGF).

6.4 Kinetic Properties of Anti-VEGF Antibodies

In certain embodiments, the anti-VEGF antibodies of the disclosure havea high binding affinity for VEGF. In specific embodiments, the anti-VEGFantibodies of the present disclosure have specific association rateconstants (k_(on) or k_(a) values), dissociation rate constants (k_(off)or k_(d) values), affinity constants (K_(A) values), dissociationconstants (K_(D) values) and/or IC₅₀ values. In various embodiments,binding constants for the interaction of the anti-VEGF antibodies withVEGF receptor can be determined using surface plasmon resonance, e.g.,according to the method disclosed in Karlsson et al., 1991, J. Immunol.Methods 145:229-240. In certain aspects, such values are selected fromthe following embodiments.

In a specific embodiment, an anti-VEGF antibody of the disclosure bindsto VEGF with a k_(on) of at least 10⁴ M⁻¹ s⁻¹, at least 5×10⁴ M⁻¹ s⁻, atleast 10⁵ M⁻¹ s⁻¹, at least 5×10⁵ M⁻¹ s⁻¹, at least 10⁶ M⁻¹ s⁻¹, atleast 5×10⁶ M⁻¹ s⁻¹, at least 10⁷ M⁻¹ s⁻¹, at least 5×10⁷ M⁻¹ s⁻¹, atleast 10⁸ M⁻¹ s⁻¹, at least 5×10⁸ M⁻¹ s⁻¹, at least 10⁹ M⁻¹ s⁻¹ or witha k_(on) of any range between any pair of the foregoing values (e.g.,5×10⁵ to 5×10⁶ M⁻¹ s⁻¹ or 10⁷ to 10⁸ M⁻¹ s⁻¹).

In another embodiment, an anti-VEGF antibody of the disclosure binds toVEGF with a k_(off) rate of 10⁻³ s⁻¹ or less, 5×10⁻⁴ s⁻¹ or less, 10⁻⁴s⁻¹ or less, 5×10⁻⁵ s⁻¹ or less, 10⁻⁵ s⁻¹ or less, 5×10⁻⁶ s⁻¹ or less,10⁻⁶ s⁻¹ or less, 5×10⁻⁷ s⁻¹ or less, 10⁻⁷ s⁻¹ or less, 5×10⁻⁸ s⁻¹ orless, 10⁻⁸ s⁻¹ or less, or with a k_(off) rate of any range between anypair of the foregoing values (e.g., 5×10⁻⁴ to 10⁻⁶ s⁻¹, or 10⁻³ to5×10⁻⁵ s⁻¹).

In another embodiment, an anti-VEGF antibody of the disclosure binds toVEGF with a K_(A) (k_(on)/k_(off)) of at least at least 10⁸ M⁻¹, atleast 5×10⁹ M⁻¹, at least 10¹⁰ M⁻¹, at least 5×10¹⁰ M⁻, 10¹¹ M⁻¹, atleast 5×10¹¹ M⁻¹, at least 10¹² M⁻¹, at least 5×10¹² M⁻¹, at least 10¹³M⁻¹, at least 5×10¹³ M⁻¹, at least 10¹⁴ M⁻¹, at least 5×10¹⁴ M⁻¹, atleast 10¹⁵ M⁻¹ or with a K_(A) of any range between any pair of theforegoing values (e.g., from 5×10⁹ M⁻¹ to 10¹¹ M⁻¹, or from 10¹¹M⁻¹ to5×10¹⁴ M⁻¹).

In other embodiments, an anti-VEGF antibody of the disclosure binds toVEGF with a K_(D) (k_(off)/k_(on)) of 10⁻⁸ M or less, 5×10⁻⁹ M or less,10⁻⁹ M or less, 5×10⁻¹⁰ M or less, 10⁻¹⁰ M or less, 5×10⁻¹¹ M or less,10⁻¹¹ M or less, 5×10⁻¹² M or less, 10⁻¹² M or less, 5×10⁻¹³ M or less,10⁻¹³ M or less, 5×10⁻¹⁴ M or less, 10⁻¹⁴ M or less, 5×10⁻¹⁵ M or less,10⁻¹⁵ M or less, or with a K_(D) of any range between any pair of theforegoing values (e.g., 5×10⁻⁹ to 5×10⁻¹² M or from 5×10^(−11 M) to5×10⁻¹³ M).

In specific embodiments, the K_(D) (k_(off)/k_(on)) value is determinedby assays well known in the art or described herein, e.g., ELISA,isothermal titration calorimetry (ITC), fluorescent polarization assayor any other biosensors such as BIAcore.

In some embodiments, an anti-VEGF antibody of the disclosure binds toVEGF and inhibits the binding of VEGF to a VEGF receptor (Flt-1 orFlk-1) at an IC₅₀ value of less than 5×10⁷ nM, less than 10⁷ nM, lessthan 5×10⁶ nM, less than 10⁶ nM, less than 5×10⁵ nM, less than 10⁵ nM,less than 5×10⁴ nM, less than 10⁴ nM, less than 5×10³ nM, less than 10³nM, less than 5×10² nM, less than 100 nM, less than 90 nM, less than 80nM, less than 70 nM, less than 65 nM, less than 60 nM, less than 50 nM,less than 40 nM, less than 30 nM, less than 25 nM, less than 20 nM, lessthan 15 nM, less than 12 nM, less than 10 nM, less than 5 nM, less than1 nM, less than 5×10⁻¹ nM, less than 10⁻¹ nM, less than 5×10⁻² nM, lessthan 10⁻² nM, less than 5×10⁻³ nM, less than 10⁻³ nM, less than 5×10⁻⁴nM, or less than 10⁻⁴ nM, or with an IC₅₀ of any range between any pairof the foregoing values (e.g., 5×10⁷ to 50 nM, or 15 nM to 5×10⁻³ nM).IC₅₀ can be measured according to methods well known in the art ordescribed herein, e.g., ELISA.

In other embodiments, an anti-VEGF antibody of the disclosure binds toVEGF and neutralizes the activity VEGF in a bioassay (e.g., ECproliferation or migration) at an IC₅₀ value of less than 5×10⁷ nM, lessthan 10⁷ nM, less than 5×10⁶ nM, less than 10⁶ nM, less than 5×10⁵ nM,less than 10⁵ nM, less than 5×10⁴ nM, less than 10⁴ nM, less than 5×10³nM, less than 10³ nM, less than 5×10² nM, less than 100 nM, less than 90nM, less than 80 nM, less than 70 nM, less than 65 nM, less than 60 nM,less than 50 nM, less than 40 nM, less than 30 nM, less than 25 nM, lessthan 20 nM, less than 15 nM, less than 12 nM, less than 10 nM, less than5 nM, less than 1 nM, less than 5×10⁻¹ nM, less than 10⁻¹ nM, less than5×10⁻² nM, less than 10⁻² nM, less than 5×10⁻³ nM, less than 10⁻³ nM,less than 5×10⁻⁴ nM, or less than 10⁻⁴ nM, or with an IC₅₀ of any rangebetween any pair of the foregoing values (e.g., 5×10⁷ to 50 nM, or 15 nMto 5×10⁻³ nM). An exemplary neutralization assay that can be used tomeasure the IC₅₀ of an anti-VEGF antibody is described in Section 6.3below.

In certain embodiments, an anti-VEGF antibody binds to VEGF and inhibitsthe binding of VEGF to Flt-1, Flk-1 or both, or inhibits VEGF activityin a VEGF neutralization assay, at an IC₅₀ value of betweenapproximately 1 pm and approximately 1 μM. In specific embodiments, ananti-VEGF antibody binds to VEGF and inhibits the binding of VEGF toFlt-1, Flk-1 or both, or inhibits VEGF activity in a VEGF neutralizationassay, at an IC₅₀ value of between 10 pM and 100 nM, between 100 pM and10 nM, between 200 pM and 5 nM, between 300 pM and 4 nM, between 500 pMand 3 nM, between 750 pM and 2 nM, between 1 nM and 20 nM, between 500pM and 40 nM, between 50 pM and 50 nM, between 250 pM and 100 nM, andbetween 100 nM and 1 μM, or with an IC₅₀ of any range between any pairof the foregoing values (e.g., 10 pM to 50 nM, or 750 pM to 2 nM).

In certain aspects of the foregoing embodiments, the IC₅₀ is measured inthe presence of VEGF at a concentration of 0.001 μM, 0.005 μM, 0.01 μM,0.05 μM, 0.1 μM, 0.5 μM, 1 μM, 10 μM, 20 μM, 30 μM, 40 μM, 50 μM, 60 μM,70 μM, 80 μM, 90 μM, 100 μM, 200 μM, 300 μM, 400 μM, 500 μM, 600 μM, 700μM, 800 μM, 900 μM, 1000 μM or at a concentration of any range betweenany pair of the foregoing values (e.g., 0.01 to 50 μM, or 10 μM to 100μM).

In certain embodiments, the kinetic properties of an antibody of thedisclosure are comparable to, or improved relative to, bevacizumab or ofranibizumab in a comparable assay. For example, in certain embodiments,an anti-VEGF antibody of the disclosure binds to VEGF with a k_(on) rateranging from approximately 0.5× to 1000× of the k_(on) of bevacizumab orof ranibizumab, for example a k_(on) of 0.5× of the k_(on) ofbevacizumab or of ranibizumab, a k_(on) of 0.75× of the k_(on) ofbevacizumab or of ranibizumab, a k_(on) of 0.9× of the k_(on) ofbevacizumab or of ranibizumab, a k_(on) of 1× of the k_(on) ofbevacizumab or of ranibizumab, a k_(on) of 1.1× of the k_(on) ofbevacizumab or of ranibizumab, a k_(on) of 1.2× of the k_(on) ofbevacizumab or of ranibizumab, a k_(on) of 1.3× of the k_(on) ofbevacizumab or of ranibizumab, a k_(on) of 1.4× of the k_(on) ofbevacizumab or of ranibizumab, a k_(on) of 1.5× of the k_(on) ofbevacizumab or of ranibizumab, a k_(on) of 1.75× of the k_(on) ofbevacizumab or of ranibizumab, a k_(on) of 2× of the k_(on) ofbevacizumab or of ranibizumab, a k_(on) of 2.25× of the k_(on) ofbevacizumab or of ranibizumab, a k_(on) of 2.5× of the k_(on) ofbevacizumab or of ranibizumab, a k_(on) of 3× of the k_(on) ofbevacizumab or of ranibizumab, a k_(on) of 4× of the k_(on) ofbevacizumab or of ranibizumab, a k_(on) of 5× of the k_(on) ofbevacizumab or of ranibizumab, a k_(on) of 7.5× of the k_(on) ofbevacizumab or of ranibizumab, a k_(on) of 10× of the k_(on) ofbevacizumab or of ranibizumab, a k_(on) of 15× of the k_(on) ofbevacizumab or of ranibizumab, a k_(on) of 20× of the k_(on) ofbevacizumab or of ranibizumab, a k_(on) of 50× of the k_(on) ofbevacizumab or of ranibizumab, a k_(on) of 75× of the k_(on) ofbevacizumab or of ranibizumab, a k_(on) of 100× of the k_(on) ofbevacizumab or of ranibizumab, a k_(on) of 150× of the k_(on) ofbevacizumab or of ranibizumab, a k_(on) of 200× of the k_(on) ofbevacizumab or of ranibizumab, a k_(on) of 200× of the k_(on) ofbevacizumab or of ranibizumab or a k_(on) ranging between any pair ofthe foregoing values, e.g., a k_(on) of 2×-75× of the k_(on) ofbevacizumab or of ranibizumab, a k_(on) of 5×-100× of the k_(on) ofbevacizumab or of ranibizumab, a k_(on) of 0.5×-1000× of the k_(on) ofbevacizumab or of ranibizumab, a k_(on) of 0.75×-200× of the k_(on) ofbevacizumab or of ranibizumab, etc.

In certain embodiments, an anti-VEGF antibody of the disclosure binds toVEGF with a k_(off) rate ranging from 0.001× to 3× of the k_(off) ofbevacizumab or of ranibizumab, for example a k_(off) of 0.002× of thek_(off) of bevacizumab or of ranibizumab, a k_(off) of 0.005× of thek_(off) of bevacizumab or of ranibizumab, a k_(off) of 0.0075× of thek_(off) of bevacizumab or of ranibizumab, a k_(off) of 0.01× of thek_(off) of bevacizumab or of ranibizumab, a k_(off) of 0.025× of thek_(off) of bevacizumab or of ranibizumab, a k_(off) of 0.05× of thek_(off) of bevacizumab or of ranibizumab, a k_(off) of 0.075× of thek_(off) of bevacizumab or of ranibizumab, a k_(off) of 0.1× of thek_(off) of bevacizumab or of ranibizumab, a k_(off) of 0.25× of thek_(off) of bevacizumab or of ranibizumab, a k_(off) of 0.5× of thek_(off) of bevacizumab or of ranibizumab, a k_(off) of 0.75× of thek_(off) of bevacizumab or of ranibizumab, a k_(off) of 0.9× of thek_(off) of bevacizumab or of ranibizumab, a k_(off) of 1× of the k_(off)of bevacizumab or of ranibizumab, a k_(off) of 1.1× of the k_(off) ofbevacizumab or of ranibizumab, a k_(off) of 1.25× of the k_(off) ofbevacizumab or of ranibizumab, a k_(off) of 1.5× of the k_(off) ofbevacizumab or of ranibizumab, a k_(off) of 1.75× of the k_(off) ofbevacizumab or of ranibizumab, a k_(off) of 4× of the k_(off) ofbevacizumab or of ranibizumab, a k_(off) of 3× of the k_(off) ofbevacizumab or of ranibizumab, a k_(off) of 2× of the k_(off) ofbevacizumab or of ranibizumab, a k_(off) of 3× of the k_(off) ofbevacizumab or of ranibizumab, or a k_(off) ranging between any pair ofthe foregoing values, e.g., a k_(off) of 0.01× to 1.1× of the k_(off) ofbevacizumab or of ranibizumab, a k_(off) of 0.05×-1.25× of the k_(off)of bevacizumab or of ranibizumab, a k_(off) of 0.1×-0.9× of the k_(off)of bevacizumab or of ranibizumab, etc.

In other embodiments, an anti-VEGF antibody of the disclosure binds toVEGF with a K_(A) (k_(on)/k_(off)) ranging from 0.25× to 1000× of theK_(A) of bevacizumab or of ranibizumab, for example a K_(A) of 0.5× ofthe K_(A) of bevacizumab or of ranibizumab, a K_(A) of 0.75× of theK_(A) of bevacizumab or of ranibizumab, a K_(A) of 1× of the K_(A) ofbevacizumab or of ranibizumab, a K_(A) of 2× of the K_(A) of bevacizumabor of ranibizumab, a K_(A) of 4× of the K_(A) of bevacizumab or ofranibizumab, a K_(A) of 10× of the K_(A) of bevacizumab or ofranibizumab, a K_(A) of 15× of the K_(A) of bevacizumab or ofranibizumab, a K_(A) of 20× of the K_(A) of bevacizumab or ofranibizumab, a K_(A) of 30× of the K_(A) of bevacizumab or ofranibizumab, a K_(A) of 40× of the K_(A) of bevacizumab or ofranibizumab, a K_(A) of 50× of the K_(A) of bevacizumab or ofranibizumab, a K_(A) of 100× of the K_(A) of bevacizumab or ofranibizumab, a K_(A) of 250× of the K_(A) of bevacizumab or ofranibizumab, a K_(A) of 500× of the K_(A) of bevacizumab or ofranibizumab, a K_(A) of 750× of the K_(A) of bevacizumab or ofranibizumab, a K_(A) of 1000× of the K_(A) of bevacizumab or ofranibizumab or a K_(A) ranging between any pair of the foregoing values,e.g., a K_(A) of 0.75×-10 5× of the K_(A) of bevacizumab or ofranibizumab, a K_(A) of 1×-100× of the K_(A) of bevacizumab or ofranibizumab, a K_(A) of 10×-20× of the K_(A) of bevacizumab or ofranibizumab, a K_(A) of 4×-50× of the K_(A) of bevacizumab or ofranibizumab, a K_(A) of 2×-20× of the K_(A) of bevacizumab or ofranibizumab, or any value or range that can be calculated from thek_(on) and k_(off) rates disclosed herein.

In other embodiments, an anti-VEGF antibody of the disclosure binds toVEGF a K_(D) (k_(off)/k_(on)) ranging from ranging from 0.001× to 10× ofthe K_(D) of bevacizumab or of ranibizumab, for example a K_(D) of0.001× of the K_(D) of bevacizumab or of ranibizumab, a K_(D) of 0.005×of the K_(D) of bevacizumab or of ranibizumab, a K_(D) of 0.01× of theK_(D) of bevacizumab or of ranibizumab, a K_(D) of 0.05× of the K_(D) ofbevacizumab or of ranibizumab, a K_(D) of 0.075× of the K_(D) ofbevacizumab or of ranibizumab, a K_(D) of 0.1× of the K_(D) ofbevacizumab or of ranibizumab, a K_(D) of 0.2× of the K_(D) ofbevacizumab or of ranibizumab, a K_(D) of 0.3× of the K_(D) ofbevacizumab or of ranibizumab, a K_(D) of 0.4× of the K_(D) ofbevacizumab or of ranibizumab, a K_(D) of 0.5× of the K_(D) ofbevacizumab or of ranibizumab, a K_(D) of 0.6× of the K_(D) ofbevacizumab or of ranibizumab, a K_(D) of 0.7× of the K_(D) ofbevacizumab or of ranibizumab, a K_(D) of 0.8× of the K_(D) ofbevacizumab or of ranibizumab, a K_(D) of 0.9× of the K_(D) ofbevacizumab or of ranibizumab, a K_(D) of 1× of the K_(D) of bevacizumabor of ranibizumab, a K_(D) of 1.5× of the K_(D) of bevacizumab or ofranibizumab, a K_(D) of 2× of the K_(D) of bevacizumab or ofranibizumab, a K_(D) of 4× of the K_(D) of bevacizumab or ofranibizumab, a K_(D) of 7.5× of the K_(D) of bevacizumab or ofranibizumab, a K_(D) of 10× of the K_(D) of bevacizumab or ofranibizumab or a K_(D) ranging between any pair of the foregoing values,e.g., a K_(D) of 0.01×-2× of the K_(D) of bevacizumab or of ranibizumab,a K_(D) of 0.1×-1.5× of the K_(D) of bevacizumab or of ranibizumab, aK_(D) of 0.7×-4× of the K_(D) of bevacizumab or of ranibizumab, a K_(D)of 0.2×-2× of the K_(D) of bevacizumab or of ranibizumab or any value orrange that can be calculated from the k_(on) and k_(off) rates disclosedherein.

In some embodiments, an anti-VEGF antibody of the disclosure binds toVEGF and inhibits the binding of VEGF to Flt-1, Flk-1 or both, orneutralize the activity of VEGF at an IC₅₀ value ranging from 0.001× to10× of the IC₅₀ of bevacizumab or of ranibizumab, for example an IC₅₀ of0.001× of the IC₅₀ of bevacizumab or of ranibizumab, an IC₅₀ of 0.005×of the IC₅₀ of bevacizumab or of ranibizumab, an IC₅₀ of 0.01× of theIC₅₀ of bevacizumab or of ranibizumab, an IC₅₀ of 0.05× of the IC₅₀ ofbevacizumab or of ranibizumab, an IC₅₀ of 0.075× of the IC₅₀ ofbevacizumab or of ranibizumab, an IC₅₀ of 0.1× of the IC₅₀ ofbevacizumab or of ranibizumab, an IC₅₀ of 0.2× of the IC₅₀ ofbevacizumab or of ranibizumab, an IC₅₀ of 0.3× of the IC₅₀ ofbevacizumab or of ranibizumab, an IC₅₀ of 0.4× of the IC₅₀ ofbevacizumab or of ranibizumab, an IC₅₀ of 0.5× of the IC₅₀ ofbevacizumab or of ranibizumab, an IC₅₀ of 0.6× of the IC₅₀ ofbevacizumab or of ranibizumab, an IC₅₀ of 0.7× of the IC₅₀ ofbevacizumab or of ranibizumab, an IC₅₀ of 0.8× of the IC₅₀ ofbevacizumab or of ranibizumab, an IC₅₀ of 0.9× of the IC₅₀ ofbevacizumab or of ranibizumab, an IC₅₀ of 1× of the IC₅₀ of bevacizumabor of ranibizumab, an IC₅₀ of 1.5× of the IC₅₀ of bevacizumab or ofranibizumab, an IC₅₀ of 2× of the IC₅₀ of bevacizumab or of ranibizumab,an IC₅₀ of 4× of the IC₅₀ of bevacizumab or of ranibizumab, an IC₅₀ of7.5× of the IC₅₀ of bevacizumab or of ranibizumab, an IC₅₀ of 10× of theIC₅₀ of bevacizumab or of ranibizumab or an IC₅₀ ranging between anypair of the foregoing values, e.g., an IC₅₀ of 0.01×-2× of the IC₅₀ ofbevacizumab or of ranibizumab, an IC₅₀ of 0.1×-1.5× of the IC₅₀ ofbevacizumab or of ranibizumab, an IC₅₀ of 0.7×-4× of the IC₅₀ ofbevacizumab or of ranibizumab, an IC₅₀ of 0.2×-2× of the IC₅₀ ofbevacizumab or of ranibizumab. In certain embodiments, a single CDRsubstitution can result in the foregoing differences in IC₅₀ as comparedto bevacizumab or ranibizumab, whereas an anti-VEGF antibody of thedisclosure can comprise such substitution and up to 16 additional CDRsubstitutions as compared to bevacizumab or ranibizumab.

6.5 Reduced Immunogenicity of Anti-VEGF Antibodies

In certain aspects, the present disclosure provides anti-VEGF antibodieshaving reduced immunogenicity as compared to bevacizumab or ranibizumab.The present disclosure provides anti-VEGF antibodies having single ormultiple amino acid substitutions in their CDRs and/or framework regionsas compared to the CDRs of bevacizumab, wherein at least onesubstitution reduces the immunogenicity of the antibody as compared tobevacizumab or ranibizumab. In certain embodiments, the reducedimmunogenicity results from one or more amino acid substitutions thatresult in eliminating or mitigating one or more T cell epitopes.

In certain aspects, the anti-VEGF antibodies of the disclosure havingreduced immunogenicity have comparable or improved biological activityas compared to bevacizumab or ranibizumab, e.g., affinity towards VEGFor neutralization of VEGF activity. Such properties can be tested, forexample, by the methods described in Section 6.3 above.

In certain embodiments, the immunogenicity of an anti-VEGF antibody ofthe disclosure is reduced relative to bevacizumab or ranibizumabantibody. Such antibodies generally have variant sequences relative tothe heavy and/or light chain variable region in regions corresponding toSEQ ID NO:25, SEQ ID NO:62 and/or SEQ ID NO:74. The antibodies willgenerally have one, two or three amino acid substitutions in one, two orall three sequences corresponding to SEQ ID NO:25, SEQ ID NO:62, and SEQID NO:74, although up to four or five substitutions in one, two or allthree regions are contemplated herein.

As used in the present disclosure, a variant with “reducedimmunogenicity” refers to an anti-VEGF antibody with a variant sequencein a region corresponding to SEQ ID NO:25, SEQ ID NO:62, and/or SEQ IDNO:74 that elicits a reduced proliferative response in peripheral bloodmononuclear cells as compared to a peptide of SEQ ID NO:25, SEQ IDNO:62, or SEQ ID NO:74, respectively. An exemplary proliferation assaythat can be used to evaluate the proliferative response is set forth inSection 7 below. The reduced proliferative response can be reflected interms of the percentage of responders, the stimulation index, or both.

In other embodiments, as compared to a peptide having the sequence ofSEQ ID NO:25, SEQ ID NO:62, or SEQ ID NO:74, the variant sequenceresults in at least 25% fewer responders, in at least 30% fewerresponders, in at least 35% fewer responders, in at least 40% fewerresponders, in at least 45% fewer responders, in at least 50% fewerresponders, in at least 60% fewer responders, in at least 65% fewerresponders, in at least 70% fewer responders, in at least 75% fewerresponders, in at least 80% fewer responders, in at least 85% fewerresponders, in at least 90% fewer responders, in at least 95% fewerresponders, 100% fewer responders, or a reduction in responders in arange between any of the foregoing values, e.g., 25%-75% fewerresponders, 50%-90% fewer responders, 60%-100% fewer responders, 70%-90%fewer responders, or the like.

In other embodiments, the variant sequence results in a stimulationindex that is at least 5% less, at least 10% less, at least 15% less, atleast 20% less, at least 25% less, at least 30% less, at least 35% less,or at least 40% less than the stimulation index elicited by a peptide ofSEQ ID NO:25, SEQ ID NO:62, or SEQ ID NO:74, respectively, or results ina stimulation index reduced by a range between any of the foregoingvalues as compared to a peptide of SEQ ID NO:25, SEQ ID NO:62, or SEQ IDNO:74, e.g., 5%-20% less, 10%-30% less, 25%-35% less, 30%-40% less, orthe like.

Exemplary embodiments of candidate anti-VEGF antibodies with reducedimmunogenicity as compared to bevacizumab or ranibizumab comprise one ormore of the CDR substitutions or combinations of substitutions set forthin FIG. 10. Optionally, anti-VEGF antibodies with reduced immunogenicityas compared to bevacizumab or ranibizumab comprise one or moreadditional substitutions, such as the CDR mutations in any of FIGS.11-17, singly or in combination.

Yet further exemplary embodiments of candidate anti-VEGF antibodies withreduced immunogenicity as compared to bevacizumab or ranibizumabcomprise one or more of the CDR substitutions or combinations ofsubstitutions set forth in FIGS. 18-20. Some preferred embodiments ofanti-VEGF antibodies with reduced immunogenicity as compared tobevacizumab or ranibizumab are provided in FIG. 23.

6.6 Antibody Conjugates

The anti-VEGF antibodies of the disclosure include antibody conjugatesthat are modified, e.g., by the covalent attachment of any type ofmolecule to the antibody, such that covalent attachment does notinterfere with binding to VEGF.

In certain aspects, an anti-VEGF antibody of the disclosure can beconjugated to an effector moiety or a label. The term “effector moiety”as used herein includes, for example, antineoplastic agents, drugs,toxins, biologically active proteins, for example enzymes, otherantibody or antibody fragments, synthetic or naturally occurringpolymers, nucleic acids (e.g., DNA and RNA), radionuclides, particularlyradioiodide, radioisotopes, chelated metals, nanoparticles and reportergroups such as fluorescent compounds or compounds which can be detectedby NMR or ESR spectroscopy.

In one example, anti-VEGF antibodies can be conjugated to an effectormoiety, such as a cytotoxic agent, a radionuclide or drug moiety tomodify a given biological response. The effector moiety can be a proteinor polypeptide, such as, for example and without limitation, a toxin(such as abrin, ricin A, Pseudomonas exotoxin, or Diphtheria toxin), asignaling molecule (such as α-interferon, β-interferon, nerve growthfactor, platelet derived growth factor or tissue plasminogen activator),a thrombotic agent or an anti-angiogenic agent (e.g., angiostatin orendostatin) or a biological response modifier such as a cytokine orgrowth factor (e.g., interleukin-1 (IL-1), interleukin-2 (IL-2),interleukin-6 (IL-6), granulocyte macrophage colony stimulating factor(GM-CSF), granulocyte colony stimulating factor (G-CSF), or nerve growthfactor (NGF)).

In another example the effector moieties can be cytotoxins or cytotoxicagents. Examples of cytotoxins and cytotoxic agents include taxol,cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin,etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin,daunorabicin, dihydroxy anthracin dione, mitoxantrone, mithramycin,actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine,tetracaine, lidocaine, propranolol, and puromycin and analogs orhomologs thereof.

Effector moieties also include, but are not limited to, antimetabolites(e.g. methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine,5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine,thioepa chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU),cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycinC5 and cis-dichlorodiamine platinum (II) (DDP) cisplatin),anthracyclines (e.g., daunorubicin (formerly daunomycin) anddoxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin),bleomycin, mithramycin, anthramycin (AMC), calicheamicins orduocarmycins), and anti-mitotic agents (e.g., vincristine andvinblastine).

Other effector moieties can include radionuclides such as, but notlimited to, ¹¹¹In and ⁹⁰Y, Lu¹⁷⁷, Bismuth²¹³, Californium²⁵², Iridium¹⁹²and Tungsten¹⁸⁸/Rhenium¹⁸⁸ and drugs such as, but not limited to,alkylphosphocholines, topoisomerase I inhibitors, taxoids and suramin.

Techniques for conjugating such effector moieties to antibodies are wellknown in the art (see, e.g., Hellstrom et al., Controlled Drug Delivery,2nd Ed., at pp. 623-53 (Robinson et al., eds., 1987)); Thorpe et al.,1982, Immunol. Rev. 62:119-58 and Dubowchik et al., 1999, Pharmacologyand Therapeutics 83:67-123).

In one example, the anti-VEGF antibody or fragment thereof is fused viaa covalent bond (e.g., a peptide bond), through the antibody'sN-terminus or C-terminus or internally, to an amino acid sequence ofanother protein (or portion thereof; for example at least a 10, 20 or 50amino acid portion of the protein). The antibody, or fragment thereof,can linked to the other protein at the N-terminus of the constant domainof the antibody. Recombinant DNA procedures can be used to create suchfusions, for example as described in WO 86/01533 and EP0392745. Inanother example the effector molecule can increase half-life in vivo,and/or enhance the delivery of an antibody across an epithelial barrierto the immune system. Examples of suitable effector molecules of thistype include polymers, albumin, albumin binding proteins or albuminbinding compounds such as those described in WO 2005/117984.

In certain aspects, an anti-VEGF antibody is conjugated to a smallmolecule toxin. In certain exemplary embodiments, an anti-VEGF antibodyof the disclosure is conjugated to a dolastatin or a dolostatin peptidicanalogs or derivatives, e.g., an auristatin (U.S. Pat. Nos. 5,635,483and 5,780,588). The dolastatin or auristatin drug moiety may be attachedto the antibody through its N (amino) terminus, C (carboxyl) terminus orinternally (WO 02/088172). Exemplary auristatin embodiments include theN-terminus linked monomethylauristatin drug moieties DE and DF, asdisclosed in U.S. Pat. No. 7,498,298, which is hereby incorporated byreference in its entirety (disclosing, e.g., linkers and methods ofpreparing monomethylvaline compounds such as MMAE and MMAF conjugated tolinkers).

In other exemplary embodiments, small molecule toxins include but arenot limited to calicheamicin, maytansine (U.S. Pat. No. 5,208,020),trichothene, and CC1065. In one embodiment of the disclosure, theantibody is conjugated to one or more maytansine molecules (e.g., about1 to about 10 maytansine molecules per antibody molecule). Maytansinemay, for example, be converted to May-SS-Me which may be reduced toMay-SH3 and reacted with an antibody (Chari et al., 1992, CancerResearch 52: 127-131) to generate a maytansinoid-antibody ormaytansinoid-Fc fusion conjugate. Structural analogues of calicheamicinthat can also be used include but are not limited to γ₁ ¹, γ₃ ¹,N-acetyl-γ₁ ¹, PSAG, and θ₁ ¹, (Hinman et al., 1993, Cancer Research53:3336-3342; Lode et al., 1998, Cancer Research 58:2925-2928; U.S. Pat.No. 5,714,586; U.S. Pat. No. 5,712,374; U.S. Pat. No. 5,264,586; U.S.Pat. No. 5,773,001).

Antibodies of the disclosure can also be conjugated to liposomes fortargeted delivery (See, e.g., Park et al., 1997, Adv. Pharmacol.40:399-435; Marty & Schwendener, 2004, Methods in Molecular Medicine109:389-401).

In one example antibodies of the present disclosure can be attached topoly(ethyleneglycol) (PEG) moieties. In one particular example theantibody is an antibody fragment and the PEG moieties can be attachedthrough any available amino acid side-chain or terminal amino acidfunctional group located in the antibody fragment, for example any freeamino, imino, thiol, hydroxyl or carboxyl group. Such amino acids canoccur naturally in the antibody fragment or can be engineered into thefragment using recombinant DNA methods. See for example U.S. Pat. No.5,219,996. Multiple sites can be used to attach two or more PEGmolecules. PEG moieties can be covalently linked through a thiol groupof at least one cysteine residue located in the antibody fragment. Wherea thiol group is used as the point of attachment, appropriatelyactivated effector moieties, for example thiol selective derivativessuch as maleimides and cysteine derivatives, can be used.

In a specific example, an anti-VEGF antibody conjugate is a modifiedFab′ fragment which is PEGylated, i.e., has PEG (poly(ethyleneglycol))covalently attached thereto, e.g., according to the method disclosed inEP0948544. See also Poly(ethyleneglycol) Chemistry, Biotechnical andBiomedical Applications, (J. Milton Harris (ed.), Plenum Press, NewYork, 1992); Poly(ethyleneglycol) Chemistry and Biological Applications,(J. Milton Harris and S. Zalipsky, eds., American Chemical Society,Washington D.C., 1997); and Bioconjugation Protein Coupling Techniquesfor the Biomedical Sciences, (M. Aslam and A. Dent, eds., GrovePublishers, New York, 1998); and Chapman, 2002, Advanced Drug DeliveryReviews 54:531-545. PEG can be attached to a cysteine in the hingeregion. In one example, a PEG-modified Fab′ fragment has a maleimidegroup covalently linked to a single thiol group in a modified hingeregion. A lysine residue can be covalently linked to the maleimide groupand to each of the amine groups on the lysine residue can be attached amethoxypoly(ethyleneglycol) polymer having a molecular weight ofapproximately 20,000 Da. The total molecular weight of the PEG attachedto the Fab′ fragment can therefore be approximately 40,000 Da.

The word “label” when used herein refers to a detectable compound orcomposition which can be conjugated directly or indirectly to ananti-VEGF antibody of the disclosure. The label can itself be detectable(e.g., radioisotope labels or fluorescent labels) or, in the case of anenzymatic label, can catalyze chemical alteration of a substratecompound or composition which is detectable. Useful fluorescent moietiesinclude, but are not limited to, fluorescein, fluoresceinisothiocyanate, rhodamine, 5-dimethylamine-1-napthalenesulfonylchloride, phycoerythrin and the like. Useful enzymatic labels include,but are not limited to, alkaline phosphatase, horseradish peroxidase,glucose oxidase and the like.

Additional anti-VEGF antibody conjugates that are useful for, interalia, diagnostic purposes, are described in Section 6.7 below.

6.7 Diagnostic Uses of Anti-VEGF Antibodies

The anti-VEGF antibodies of the disclosure, including those antibodiesthat have been modified, e.g., by biotinylation, horseradish peroxidase,or any other detectable moiety (including those described in Section6.6), can be advantageously used for diagnostic purposes.

In particular, the anti-VEGF antibodies can be used, for example, butnot limited to, to purify or detect VEGF, including both in vitro and invivo diagnostic methods. For example, the antibodies have use inimmunoassays for qualitatively and quantitatively measuring levels ofVEGF in biological samples. See, e.g., Harlow et al., Antibodies: ALaboratory Manual, Second Edition (Cold Spring Harbor Laboratory Press,1988), which is incorporated by reference herein in its entirety.

The present disclosure further encompasses antibodies or fragmentsthereof conjugated to a diagnostic agent. The antibodies can be useddiagnostically, for example, to detect expression of a target ofinterest in specific cells, tissues, or serum; or to monitor thedevelopment or progression of an immunologic response as part of aclinical testing procedure to, e.g., determine the efficacy of a giventreatment regimen. Detection can be facilitated by coupling the antibodyto a detectable substance. Examples of detectable substances includevarious enzymes, prosthetic groups, fluorescent materials, luminescentmaterials, bioluminescent materials, radioactive materials, positronemitting metals using various positron emission tomographies, andnonradioactive paramagnetic metal ions. The detectable substance can becoupled or conjugated either directly to the antibody (or fragmentthereof) or indirectly, through an intermediate (such as, for example, alinker known in the art) using techniques known in the art. Examples ofenzymatic labels include luciferases (e.g., firefly luciferase andbacterial luciferase; U.S. Pat. No. 4,737,456), luciferin,2,3-dihydrophthalazinediones, malate dehydrogenase, urease, peroxidasesuch as horseradish peroxidase (HRPO), alkaline phosphatase,β-galactosidase, acetylcholinesterase, glucoamylase, lysozyme,saccharide oxidases (e.g., glucose oxidase, galactose oxidase, andglucose-6-phosphate dehydrogenase), heterocyclic oxidases (such asuricase and xanthine oxidase), lactoperoxidase, microperoxidase, and thelike. Examples of suitable prosthetic group complexes includestreptavidin/biotin and avidin/biotin; examples of suitable fluorescentmaterials include umbelliferone, fluorescein, fluoresceinisothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansylchloride or phycoerythrin; an example of a luminescent material includesluminol; examples of bioluminescent materials include luciferase,luciferin, and aequorin; and examples of suitable radioactive materialinclude ¹²⁵I, ¹³¹I, ¹¹¹In or ⁹⁹Tc.

The disclosure provides for the detection of expression of VEGF,comprising contacting a biological sample (cells, tissue, or body fluidof an individual) using one or more anti-VEGF antibodies of thedisclosure (optionally conjugated to detectable moiety), and detectingwhether or not the sample is positive for VEGF expression, or whetherthe sample has altered (e.g., reduced or increased) expression ascompared to a control sample.

Diseases that can be diagnosed using the present methods include, butare not limited to, the diseases described herein. In certainembodiments, the tissue or body fluid is peripheral blood, peripheralblood leukocytes, biopsy tissues such as lung or skin biopsies, andtissue.

6.8 Therapeutic Methods Using Anti-VEGF Antibodies

6.8.1 Clinical Benefits

The anti-VEGF antibodies of the disclosure can be used to treat variousneoplasms or non-neoplastic conditions characterized by pathologicalangiogenesis.

The antibodies of the disclosure are useful in the treatment of tumorsin which angiogenesis plays an important role in tumor growth, includingcancers and benign tumors. Examples of cancer to be treated hereininclude, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma,and leukemia. More particular examples of such cancers include squamouscell cancer, lung cancer (including small-cell lung cancer, non-smallcell lung cancer, adenocarcinoma of the lung, and squamous carcinoma ofthe lung), cancer of the peritoneum, hepatocellular cancer, gastric orstomach cancer (including gastrointestinal cancer), pancreatic cancer,glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladdercancer, hepatoma, breast cancer, colon cancer, colorectal cancer,endometrial or uterine carcinoma, salivary gland carcinoma, kidney orrenal cancer, liver cancer, prostate cancer, vulval cancer, thyroidcancer, hepatic carcinoma and various types of head and neck cancer, aswell as B-cell lymphoma (including low grade/follicular non-Hodgkin'slymphoma (NHL); small lymphocytic (SL) NHL; intermediategrade/follicular NHL; intermediate grade diffuse NHL; high gradeimmunoblastic NHL; high grade lymphoblastic NHL; high grade smallnon-cleaved cell NHL; bulky disease NHL; mantle cell lymphoma;AIDS-related lymphoma; and Waldenstrom's Macroglobulinemia); chroniclymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); Hairycell leukemia; chronic myeloblastic leukemia; and post-transplantlymphoproliferative disorder (PTLD), as well as abnormal vascularproliferation associated with phakomatoses, edema (such as thatassociated with brain tumors), and Meigs' syndrome. More particularly,cancers that are amenable to treatment by the antibodies of thedisclosure include breast cancer, colorectal cancer, rectal cancer,non-small cell lung cancer, non-Hodgkins lymphoma (NHL), renal cellcancer, prostate cancer, liver cancer, pancreatic cancer, soft-tissuesarcoma, kaposi's sarcoma, carcinoid carcinoma, head and neck cancer,melanoma, ovarian cancer, mesothelioma, and multiple myeloma. In someembodiments, the anti-VEGF antibodies of the disclosure are used totreat colorectal cancer in a human patient.

The present disclosure encompasses anti-angiogenic therapy, a cancertreatment strategy aimed at inhibiting the development of tumor bloodvessels required for providing nutrients to support tumor growth.Because angiogenesis is involved in both primary tumor growth andmetastasis, the antiangiogenic treatment provided by the disclosure iscapable of inhibiting the neoplastic growth of tumor at the primary siteas well as preventing metastasis of tumors at the secondary sites.

Non-neoplastic conditions that are amenable to treatment with theantibodies of the disclosure include retinal diseases (e.g., age-relatedmacular degeneration) and immune and inflammatory diseases (e.g.,rheumatoid arthritis, psoriasis, atherosclerosis, diabetic and otherproliferative retinopathies including retinopathy of prematurity,retrolental fibroplasia, neovascular glaucoma, age-related maculardegeneration, thyroid hyperplasias (including Grave's disease), cornealand other tissue transplantation, chronic inflammation, lunginflammation, nephrotic syndrome, preeclampsia, ascites, pericardialeffusion (such as that associated with pericarditis), and pleuraleffusion).

Accordingly, the present disclosure provides methods of treating any ofthe foregoing diseases in a patient in need thereof, comprising:administering to the patient an anti-VEGF antibody of the disclosure.Optionally, said administration is repeated, e.g., after one day, twodays, three days, five days, one week, two weeks, three weeks, onemonth, five weeks, six weeks, seven weeks, eight weeks, two months orthree months. The repeated administration can be at the same dose or ata different dose. The administration can be repeated once, twice, threetimes, four times, five times, six times, seven times, eight times, ninetimes, ten times, or more. For example, according to certain dosageregimens a patient receives anti-VEGF therapy for a prolonged period oftime, e.g., 6 months, 1 year or more. The amount of anti-VEGF antibodyadministered to the patient is in certain embodiments a therapeuticallyeffective amount. As used herein, a “therapeutically effective” amountof VEGF antibody can be administered as a single dose or over the courseof a therapeutic regimen, e.g., over the course of a week, two weeks,three weeks, one month, three months, six months, one year, or longer.Exemplary therapeutic regimens are described in Section 6.11 below.

According to the present disclosure, treatment of a disease encompassesthe treatment of patients already diagnosed as having any form of thedisease at any clinical stage or manifestation; the delay of the onsetor evolution or aggravation or deterioration of the symptoms or signs ofthe disease; and/or preventing and/or reducing the severity of thedisease.

A “subject” or “patient” to whom the anti-VEGF antibody of thedisclosure is administered is preferably a mammal such as a non-primate(e.g., cow, pig, horse, cat, dog, rat, etc.) or a primate (e.g., monkeyor human). In certain embodiments, the subject or patient is a human. Incertain aspects, the human is a pediatric patient. In other aspects, thehuman is an adult patient.

6.9 Pharmaceutical Compositions and Routes of Administration

Compositions comprising an anti-VEGF antibody of the disclosure and,optionally one or more additional therapeutic agents, such as thecombination therapeutic agents described in Section 6.10 below, areprovided herein. The compositions will usually be supplied as part of asterile, pharmaceutical composition that will normally include apharmaceutically acceptable carrier. This composition can be in anysuitable form (depending upon the desired method of administering it toa patient).

The anti-VEGF antibodies of the disclosure can be administered to apatient by a variety of routes such as orally, transdermally,subcutaneously, intranasally, intravenously, intramuscularly,intraocularly, topically, intrathecally and intracerebroventricularly.The most suitable route for administration in any given case will dependon the particular antibody, the subject, and the nature and severity ofthe disease and the physical condition of the subject.

For treatment of indications other than retinal diseases, the effectivedose of an anti-VEGF antibody of the disclosure can range from about 0.1to about 75 mg/kg per single (e.g., bolus) administration, multipleadministrations or continuous administration, or to achieve a serumconcentration of 0.01-5000 μg/mL serum concentration per single (e.g.,bolus) administration, multiple administrations or continuousadministration, or any effective range or value therein depending on thecondition being treated, the route of administration and the age, weightand condition of the subject. In certain embodiments, e.g. for thetreatment of cancer, each dose can range from about 0.1 mg to about 50mg per kilogram of body weight, for example from about 3 mg to about 25mg per kilogram body weight. The antibody can be formulated as anaqueous solution and administered by subcutaneous injection.

For treatment of retinal diseases (e.g., age-related maculardegeneration (AMD), the dosage suitably results in aqueous humorconcentration of the anti-VEGF antibody the injected eye of 1-50 μg/mL.For treatment of AMD, each dose can be from 0.1 mg to about 1 mg, forexample from about 0.25 to about 0.5 mg. The antibody can be formulatedas an aqueous solution and administered by intravitreal injection.

Pharmaceutical compositions can be conveniently presented in unit doseforms containing a predetermined amount of an anti-VEGF antibody of thedisclosure per dose. Such a unit can contain for example but withoutlimitation 0.1 mg to 5 g, for example 1 mg to 1 g, or 10 to 50 mg.Pharmaceutically acceptable carriers for use in the disclosure can takea wide variety of forms depending, e.g., on the condition to be treatedor route of administration.

Therapeutic formulations of the anti-VEGF antibodies of the disclosurecan be prepared for storage as lyophilized formulations or aqueoussolutions by mixing the antibody having the desired degree of puritywith optional pharmaceutically-acceptable carriers, excipients orstabilizers typically employed in the art (all of which are referred toherein as “carriers”), i.e., buffering agents, stabilizing agents,preservatives, isotonifiers, non-ionic detergents, antioxidants, andother miscellaneous additives. See, Remington's Pharmaceutical Sciences,16th edition (Osol, ed. 1980). Such additives must be nontoxic to therecipients at the dosages and concentrations employed.

Buffering agents help to maintain the pH in the range which approximatesphysiological conditions. They can be present at concentration rangingfrom about 2 mM to about 50 mM. Suitable buffering agents for use withthe present disclosure include both organic and inorganic acids andsalts thereof such as citrate buffers (e.g., monosodium citrate-disodiumcitrate mixture, citric acid-trisodium citrate mixture, citricacid-monosodium citrate mixture, etc.), succinate buffers (e.g.,succinic acid-monosodium succinate mixture, succinic acid-sodiumhydroxide mixture, succinic acid-disodium succinate mixture, etc.),tartrate buffers (e.g., tartaric acid-sodium tartrate mixture, tartaricacid-potassium tartrate mixture, tartaric acid-sodium hydroxide mixture,etc.), fumarate buffers (e.g., fumaric acid-monosodium fumarate mixture,fumaric acid-disodium fumarate mixture, monosodium fumarate-disodiumfumarate mixture, etc.), gluconate buffers (e.g., gluconic acid-sodiumglyconate mixture, gluconic acid-sodium hydroxide mixture, gluconicacid-potassium glyuconate mixture, etc.), oxalate buffer (e.g., oxalicacid-sodium oxalate mixture, oxalic acid-sodium hydroxide mixture,oxalic acid-potassium oxalate mixture, etc.), lactate buffers (e.g.,lactic acid-sodium lactate mixture, lactic acid-sodium hydroxidemixture, lactic acid-potassium lactate mixture, etc.) and acetatebuffers (e.g., acetic acid-sodium acetate mixture, acetic acid-sodiumhydroxide mixture, etc.). Additionally, phosphate buffers, histidinebuffers and trimethylamine salts such as Tris can be used.

Preservatives can be added to retard microbial growth, and can be addedin amounts ranging from 0.2%-1% (w/v). Suitable preservatives for usewith the present disclosure include phenol, benzyl alcohol, meta-cresol,methyl paraben, propyl paraben, octadecyldimethylbenzyl ammoniumchloride, benzalconium halides (e.g., chloride, bromide, and iodide),hexamethonium chloride, and alkyl parabens such as methyl or propylparaben, catechol, resorcinol, cyclohexanol, and 3-pentanol.Isotonicifiers sometimes known as “stabilizers” can be added to ensureisotonicity of liquid compositions of the present disclosure and includepolhydric sugar alcohols, for example trihydric or higher sugaralcohols, such as glycerin, erythritol, arabitol, xylitol, sorbitol andmannitol. Stabilizers refer to a broad category of excipients which canrange in function from a bulking agent to an additive which solubilizesthe therapeutic agent or helps to prevent denaturation or adherence tothe container wall. Typical stabilizers can be polyhydric sugar alcohols(enumerated above); amino acids such as arginine, lysine, glycine,glutamine, asparagine, histidine, alanine, ornithine, L-leucine,2-phenylalanine, glutamic acid, threonine, etc., organic sugars or sugaralcohols, such as lactose, trehalose, stachyose, mannitol, sorbitol,xylitol, ribitol, myoinisitol, galactitol, glycerol and the like,including cyclitols such as inositol; polyethylene glycol; amino acidpolymers; sulfur containing reducing agents, such as urea, glutathione,thioctic acid, sodium thioglycolate, thioglycerol, α-monothioglyceroland sodium thio sulfate; low molecular weight polypeptides (e.g.,peptides of 10 residues or fewer); proteins such as human serum albumin,bovine serum albumin, gelatin or immunoglobulins; hydrophylic polymers,such as polyvinylpyrrolidone monosaccharides, such as xylose, mannose,fructose, glucose; disaccharides such as lactose, maltose, sucrose andtrisaccacharides such as raffinose; and polysaccharides such as dextran.Stabilizers can be present in the range from 0.1 to 10,000 weights perpart of weight active protein.

Non-ionic surfactants or detergents (also known as “wetting agents”) canbe added to help solubilize the therapeutic agent as well as to protectthe therapeutic protein against agitation-induced aggregation, whichalso permits the formulation to be exposed to shear surface stressedwithout causing denaturation of the protein. Suitable non-ionicsurfactants include polysorbates (20, 80, etc.), polyoxamers (184, 188etc.), Pluronic polyols, polyoxyethylene sorbitan monoethers (TWEEN®-20,TWEEN®-80, etc.). Non-ionic surfactants can be present in a range ofabout 0.05 mg/mL to about 1.0 mg/mL, for example about 0.07 mg/mL toabout 0.2 mg/mL.

Additional miscellaneous excipients include bulking agents (e.g.,starch), chelating agents (e.g., EDTA), antioxidants (e.g., ascorbicacid, methionine, vitamin E), and cosolvents.

The formulation herein can also contain a combination therapeutic agentin addition to the anti-VEGF antibody of the disclosure. Examples ofsuitable combination therapeutic agents are provided in Section 6.10below.

The dosing schedule for subcutaneous administration can vary from onceevery six months to daily depending on a number of clinical factors,including the type of disease, severity of disease, and the patient'ssensitivity to the anti-VEGF antibody.

The dosage of an anti-VEGF antibody of the disclosure to be administeredof will vary according to the particular antibody, the type of disease(e.g., cancer, inflammatory, etc.), the subject, and the severity of thedisease, the physical condition of the subject, the therapeutic regimen(e.g., whether a combination therapeutic agent is used), and theselected route of administration; the appropriate dosage can be readilydetermined by a person skilled in the art.

It will be recognized by one of skill in the art that the optimalquantity and spacing of individual dosages of an anti-VEGF antibody ofthe disclosure will be determined by the nature and extent of thecondition being treated, the form, route and site of administration, andthe age and condition of the particular subject being treated, and thata physician will ultimately determine appropriate dosages to be used.This dosage can be repeated as often as appropriate. If side effectsdevelop the amount and/or frequency of the dosage can be altered orreduced, in accordance with normal clinical practice.

6.10 Combination Therapy

Described below are combinatorial methods in which the anti-VEGFantibodies of the disclosure can be utilized. The combinatorial methodsof the disclosure involve the administration of at least two agents to apatient, the first of which is an anti-VEGF antibody of the disclosure,and the second of which is a combination therapeutic agent. Theanti-VEGF antibody and the combination therapeutic agent can beadministered simultaneously, sequentially or separately.

The combinatorial therapy methods of the present disclosure can resultin a greater than additive effect, providing therapeutic benefits whereneither the anti-VEGF antibody or combination therapeutic agentadministered in an amount that is alone therapeutically effective.

In the present methods, the anti-VEGF antibody of the disclosure and thecombination therapeutic agent can be administered concurrently, eithersimultaneously or successively. As used herein, the anti-VEGF antibodyof the disclosure and the combination therapeutic agent are said to beadministered successively if they are administered to the patient on thesame day, for example during the same patient visit. Successiveadministration can occur 1, 2, 3, 4, 5, 6, 7 or 8 hours apart. Incontrast, the anti-VEGF antibody of the disclosure and the combinationtherapeutic agent are said to be administered separately if they areadministered to the patient on the different days, for example, theanti-VEGF antibody of the disclosure and the combination therapeuticagent can be administered at a 1-day, 2-day or 3-day, one-week, 2-weekor monthly intervals. In the methods of the present disclosure,administration of the anti-VEGF antibody of the disclosure can precedeor follow administration of the combination therapeutic agent.

As a non-limiting example, the anti-VEGF antibody of the disclosure andcombination therapeutic agent can be administered concurrently for aperiod of time, followed by a second period of time in which theadministration of the anti-VEGF antibody of the disclosure and thecombination therapeutic agent is alternated.

Because of the potentially synergistic effects of administering ananti-VEGF antibody of the disclosure and a combination therapeuticagent, such agents can be administered in amounts that, if one or bothof the agents is administered alone, is/are not therapeuticallyeffective.

In certain aspects, the combination therapeutic agent is ananti-angiogenic agent, an anti-rheumatic drug, an anti-inflammatoryagent, a chemotherapeutic agent, a radiotherapeutic, animmunosuppressive agent, or a cytotoxic drug.

It is contemplated that when used to treat various diseases, theanti-VEGF antibodies of the disclosure can be combined with othertherapeutic agents suitable for the same or similar diseases. When usedfor treating cancer, antibodies of the present disclosure may be used incombination with conventional cancer therapies, such as surgery,radiotherapy, chemotherapy or combinations thereof.

In some other aspects, other therapeutic agents useful for combinationtumor therapy with the antibody of the disclosure include antagonists,e.g., antibodies, of other factors that are involved in tumor growth,such as EGFR, ErbB2 (also known as Her2), ErbB3, ErbB4, or TNF-α.

Sometimes, for treatment of cancers and immune diseases, it may bebeneficial to also administer one or more cytokines to the patient. In apreferred embodiment, the VEGF antibody is co-administered with a growthinhibitory agent.

Suitable dosages for the growth inhibitory agent are those presentlyused and may be lowered due to the combined action (synergy) of thegrowth inhibitory agent and anti-VEGF antibody.

For treatment of cancers, immune diseases and retinal diseases,anti-inflammatory agents can suitably be used in combination with theanti-VEGF antibodies of the disclosure. Anti-inflammatory agentsinclude, but are not limited to, acetaminophen, diphenhydramine,meperidine, dexamethasone, pentasa, mesalazine, asacol, codeinephosphate, benorylate, fenbufen, naprosyn, diclofenac, etodolac andindomethacin, aspirin and ibuprofen.

For treatment of cancers, chemotherapeutic agents can suitably be usedin combination with the anti-VEGF antibodies of the disclosure.Chemotherapeutic agents include, but are not limited to, radioactivemolecules, toxins, also referred to as cytotoxins or cytotoxic agents,which includes any agent that is detrimental to the viability of cells,agents, and liposomes or other vesicles containing chemotherapeuticcompounds. Examples of suitable chemotherapeutic agents include but arenot limited to 1-dehydrotestosterone, 5-fluorouracil decarbazine,6-mercaptopurine, 6-thioguanine, actinomycin D, adriamycin, aldesleukin,an anti-α5β1 integrin antibody, alkylating agents, allopurinol sodium,altretamine, amifostine, anastrozole, anthramycin (AMC)), anti-mitoticagents, cis-dichlorodiamine platinum (II) (DDP) cisplatin, diaminodichloro platinum, anthracyclines, antibiotics, antimetabolites,asparaginase, BCG live (intravesical), betamethasone sodium phosphateand betamethasone acetate, bicalutamide, bleomycin sulfate, busulfan,calcium leucouorin, calicheamicin, capecitabine, carboplatin, lomustine(CCNU), carmustine (BSNU), Chlorambucil, Cisplatin, Cladribine,Colchicin, conjugated estrogens, Cyclophosphamide, Cyclothosphamide,Cytarabine, Cytarabine, cytochalasin B, Cytoxan, Dacarbazine,Dactinomycin, dactinomycin (formerly actinomycin), daunirubicin HCL,daunorucbicin citrate, denileukin diftitox, Dexrazoxane,Dibromomannitol, dihydroxy anthracin dione, Docetaxel, dolasetronmesylate, doxorubicin HCL, dronabinol, E. coli L-asparaginase,eolociximab, emetine, epoetin-α, Erwinia L-asparaginase, esterifiedestrogens, estradiol, estramustine phosphate sodium, ethidium bromide,ethinyl estradiol, etidronate, etoposide citrororum factor, etoposidephosphate, filgrastim, floxuridine, fluconazole, fludarabine phosphate,fluorouracil, flutamide, folinic acid, gemcitabine HCL, glucocorticoids,goserelin acetate, gramicidin D, granisetron HCL, hydroxyurea,idarubicin HCL, ifosfamide, interferon α-2b, irinotecan HCL, letrozole,leucovorin calcium, leuprolide acetate, levamisole HCL, lidocaine,lomustine, maytansinoid, mechlorethamine HCL, medroxyprogesteroneacetate, megestrol acetate, melphalan HCL, mercaptipurine, mesna,methotrexate, methyltestosterone, mithramycin, mitomycin C, mitotane,mitoxantrone, nilutamide, octreotide acetate, ondansetron HCL,paclitaxel, pamidronate disodium, pentostatin, pilocarpine HCL,plimycin, polifeprosan 20 with carmustine implant, porfimer sodium,procaine, procarbazine HCL, propranolol, rituximab, sargramostim,streptozotocin, tamoxifen, taxol, teniposide, tenoposide, testolactone,tetracaine, thioepa chlorambucil, thioguanine, thiotepa, topotecan HCL,toremifene citrate, trastuzumab, tretinoin, valrubicin, vinblastinesulfate, vincristine sulfate, and vinorelbine tartrate.

Any anti-angiogenic agent can be used in conjunction with the anti-VEGFantibodies of the disclosure, including those listed by Carmeliet andJain, 2000, Nature 407:249-257. In certain embodiments, theanti-angiogenic agent is another VEGF antagonist or a VEGF receptorantagonist such as VEGF variants, soluble VEGF receptor fragments,aptamers capable of blocking VEGF or VEGFR, neutralizing anti-VEGFRantibodies, low molecule weight inhibitors of VEGFR tyrosine kinases andany combinations thereof. Alternatively, or in addition, two or moreanti-VEGF antibodies may be co-administered to the patient.

In certain embodiments, hormone therapy can be used in conjunction withanti-VEGF antibodies of the disclosure. In some embodiments, the hormonetherapy includes one or more agents that inhibit estrogen and/orprogesterone from promoting cancer cell growth, e.g., a selectiveestrogen-receptor modulator such as tamoxifen, an aromatase inhibitorsuch as anastrozole (Arimidex®) or letrozole (Femara), an aromataseinactivator such as exemestane (Aromasin®), or an agent that inhibitsestrogen production such as goserelin (Zoladex). In other embodiments,the hormone therapy is one or more agents that inhibit production ofhormones from the ovaries.

In some aspects, an anti-VEGF antibody can be used in conjunction with asmall molecule protein tyrosine kinase (PTK) inhibitor. In someembodiments, the PTK inhibitor is specific for a VEGF receptor tyrosinekinase. In other embodiments, the PTK inhibitor binds to more than oneof the VEGF receptor family of tyrosine kinases (e.g., VEGFR-1,VEGFR-2). In other embodiments, protein tyrosine kinase inhibitorsuseful in the compositions and methods of the invention include PTKinhibitors that do not bind selectively to the VEGF family of receptortyrosine kinases, but also bind to the tyrosine kinase domains of otherfamilies of proteins such as HER2, HER3, HER4, PDGFR, and/or Raf.

In some embodiments, the tyrosine kinase is a receptor tyrosine kinase,i.e., is an intra-cellular domain of a larger protein that has anextra-cellular ligand binding domain and is activated by the binding ofone or more ligands. In certain embodiments, the protein tyrosine kinaseis a non-receptor tyrosine kinase. PTK inhibitors for use in the methodsof the present disclosure include, but are not limited to, gefitinib(ZD-1839, Iressa®), erlotinib (OSI-1774, Tarceva™), canertinib(CI-1033), vandetanib (ZD6474, Zactima®), tyrphostin AG-825 (CAS149092-50-2), lapatinib (GW-572016), sorafenib (BAY43-9006), AG-494 (CAS133550-35-3), RG-13022 (CAS 149286-90-8), RG-14620 (CAS 136831-49-7),BIBW 2992 (Tovok), tyrphostin 9 (CAS 136831-49-7), tyrphostin 23 (CAS118409-57-7), tyrphostin 25 (CAS 118409-58-8), tyrphostin 46 (CAS122520-85-8), tyrphostin 47 (CAS 122520-86-9), tyrphostin 53 (CAS122520-90-5), butein(1-(2,4-dihydroxyphenyl)-3-(3,4-dihydroxyphenyl)-2-propen-1-one2′,3,4,4′-Tetrahydroxychalcone; CAS 487-52-5), curcumin((E,E)-1,7-bis(4-Hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione; CAS458-37-7),N4-(1-Benzyl-1H-indazol-5-yl)-N6,N6-dimethyl-pyrido-[3,4-d]-pyrimidine-4,6-diamine(202272-68-2), AG-1478, AG-879, Cyclopropanecarboxylicacid-(3-(6-(3-trifluoromethyl-phenylamino)-pyrimidin-4-ylamino)-phenyl)-amide(CAS 879127-07-8),N8-(3-Chloro-4-fluorophenyl)-N2-(1-methylpiperidin-4-yl)-pyrimido[5,4-d]pyrimidine-2,8-diamine,2HCl (CAS 196612-93-8), 4-(4-Benzyloxyanilino)-6,7-dimethoxyquinazoline(CAS 179248-61-4),N-(4-((3-Chloro-4-fluorophenyl)amino)pyrido[3,4-d]pyrimidin-6-yl)-2-butynamide(CAS 881001-19-0), EKB-569, HKI-272, and HKI-357.

In a specific embodiment, an anti-VEGF antibody of the disclosure isused in combination with intravenous 5-fluorouracil-based chemotherapy.This combination is suitable for, inter alia, first- or second-linetreatment of patients with metastatic carcinoma of the colon or rectum.

In another specific embodiment, an anti-VEGF antibody of the disclosureis used in combination with carboplatin and paclitaxel. This combinationis suitable for, inter alia, first-line treatment of patients withunresectable, locally advanced, recurrent or metastatic non-squamous,non-small cell lung cancer.

In yet another specific embodiment, an anti-VEGF antibody of thedisclosure is used in combination with paclitaxel. This combination issuitable for, inter alia, treatment of patients who have not receivedchemotherapy for metastatic HER2-negative breast cancer.

For treatment of retinal diseases, the anti-VEGF antibodies of thedisclosure can be used in combination with E10030, ananti-platelet-derived growth factor (PDGF) pegylated aptamer; withARC1905, a pegylated aptamer targeting the C5 component of thecomplement cascade; and volociximab, a monoclonal antibody targeting theα5β1 integrin transmembrane receptor; photodynamic therapy withVisudyne® (PDT); or Macugen®, an aptamer (pegaptanib sodium).

6.11 Therapeutic Regimens

The present disclosure provides therapeutic regimens involving theadministration of the anti-VEGF antibodies of the disclosure. Thetherapeutic regimen will vary depending on the patient's age, weight,and disease condition. The therapeutic regimen can continue for 2 weeksto indefinitely. In specific embodiments, the therapeutic regimen iscontinued for 2 weeks to 6 months, from 3 months to 5 years, from 6months to 1 or 2 years, from 8 months to 18 months, or the like. Thetherapeutic regimen can be a non-variable dose regimen or amultiple-variable dose regimen.

For the dosage exemplary regimens described below, the anti-VEGFantibody can be administered as a sterile, preservative-free solutionfor subcutaneous administration.

For treatment of metastatic colorectal cancer, an anti-VEGF antibody ofthe disclosure is administered intravenously at a dose of 0.5-15 mg/kgevery 2 weeks with bolus-IFL (irinotecan, 5-fluorouracil and leucovorinregimen). In specific embodiments, the dose is 1-4 mg/kg, 2-6 mg/kg,0.5-3 mg/kg, 1-10 mg/kg, 3-4.8 mg/kg or 1-4.5 mg/kg every two weeks withbolus-IFL.

In another embodiment for treatment of metastatic colorectal cancer, ananti-VEGF antibody of the disclosure is administered intravenously at adose of 1-30 mg/kg every 2 weeks with FOLFOX4 (oxaliplatin, leucovorin,and fluorouracil regimen). In specific embodiments, the dose is 2-9mg/kg, 3-12 mg/kg, 1-7.5 mg/kg, 2-20 mg/kg, 6-9.75 mg/kg or 4-9.5 mg/kgevery two weeks with FOLFOX4.

For treatment of non-squamous non-small cell lung cancer, an anti-VEGFantibody of the disclosure is administered intravenously at a dose of2-40 mg/kg every three weeks with carboplatin/paclitaxel. In specificembodiments, the dose is 5-14 mg/kg, 4-20 mg/kg, 10-17.5 mg/kg, 7-14mg/kg, 10-30 mg/kg or 3-30 mg/kg every three weeks withcarboplatin/paclitaxel.

For treatment of metastatic breast cancer, an anti-VEGF antibody of thedisclosure is administered intravenously at a dose of 0.5-20 mg/kg everytwo weeks with paclitaxel. In specific embodiments, the dose is 1-4mg/kg, 2-6 mg/kg, 0.5-3 mg/kg, 1-10 mg/kg, 3-4.8 mg/kg or 1-4.5 mg/kgevery two weeks with paclitaxel.

For treatment of metastatic breast cancer, an anti-VEGF antibody of thedisclosure is administered intravenously at a dose of 0.5-20 mg/kg everytwo weeks as monotherapy. In specific embodiments, the dose is 1-4mg/kg, 2-6 mg/kg, 0.5-3 mg/kg, 1-10 mg/kg, 3-4.8 mg/kg or 1-4.5 mg/kgevery two weeks as monotherapy.

For treatment of age-related macular degeneration (AMD), an anti-VEGFantibody of the disclosure is administered at a dose of 0.1-1 mg byintravitreal injection once a month (approximately 28 days). In specificembodiments, the dose is 0.1-0.4 mg, 0.2-0.6 mg, 0.1-0.25 mg, 0.25-0.5mg, 0.25-0.75 mg, or 0.3-0.45 mg by intravitreal injection once a month(approximately 28 days). In a specific embodiment, a patient treatedwith an anti-VEGF antibody of the disclosure has wet AMD. In anotherspecific embodiment, a patient has dry AMD.

6.12 Diagnostic and Pharmaceutical Kits

Encompassed by the present disclosure are pharmaceutical kits containingthe anti-VEGF antibodies (including antibody conjugates) of thedisclosure. The pharmaceutical kit is a package comprising the anti-VEGFantibody of the disclosure (e.g., either in lyophilized form or as anaqueous solution) and one or more of the following:

-   -   A combination therapeutic agent, for example as described in        Section 6.10 above;    -   A device for administering the anti-VEGF antibody, for example a        pen, needle and/or syringe; and    -   Pharmaceutical grade water or buffer to resuspend the antibody        if the antibody is in lyophilized form.

In certain aspects, each unit dose of the anti-VEGF antibody is packagedseparately, and a kit can contain one or more unit doses (e.g., two unitdoses, three unit doses, four unit doses, five unit doses, eight unitdoses, ten unit doses, or more). In a specific embodiment, the one ormore unit doses are each housed in a syringe or pen.

Diagnostic kits containing the anti-VEGF antibodies (including antibodyconjugates) of the disclosure are also encompassed herein. Thediagnostic kit is a package comprising the anti-VEGF antibody of thedisclosure (e.g., either in lyophilized form or as an aqueous solution)and one or more reagents useful for performing a diagnostic assay. Wherethe anti-VEGF antibody is labeled with an enzyme, the kit can includesubstrates and cofactors required by the enzyme (e.g., a substrateprecursor which provides the detectable chromophore or fluorophore). Inaddition, other additives can be included, such as stabilizers, buffers(e.g., a block buffer or lysis buffer), and the like. In certainembodiments, the anti-VEGF antibody included in a diagnostic kit isimmobilized on a solid surface, or a solid surface (e.g., a slide) onwhich the antibody can be immobilized is included in the kit. Therelative amounts of the various reagents can be varied widely to providefor concentrations in solution of the reagents which substantiallyoptimize the sensitivity of the assay. In a specific embodiment, theantibody and one or more reagents can be provided (individually orcombined) as dry powders, usually lyophilized, including excipientswhich on dissolution will provide a reagent solution having theappropriate concentration.

7. EXAMPLE 1 Identification of T-Cell Epitopes of Bevacizumab

7.1 Materials & Methods

7.1.1 Peptides

Peptides were synthesized using a multi-pin format by Mimotopes(Adelaide, Australia). The sequences of the bevacizumab light and heavychain V regions were synthesized as 15-mer peptides overlapping by 12amino acids (FIGS. 7 and 8) for a total of 69 peptides. Peptides arrivedlyophilized and were re-suspended in DMSO (Sigma-Aldrich) atapproximately 1-2 mg/mL. Stock peptides were kept frozen at −20° C.

7.1.2 Human Peripheral Blood Mononuclear Cells

Community donor buffy coat products were purchased from the StanfordBlood Center, Palo Alto, Calif. Buffy coat material was diluted 1:1 v:vwith DPBS containing no calcium or magnesium. Diluted buffy coatmaterial (25-35 mls) was underlayed in 50 ml conical centrifuge tubes(Sarsted or Costar) with 12.5 mls of FicollPaque-PLUS (GE Healthcare).The samples were centrifuged at 900 g for 30 minutes at roomtemperature. Peripheral blood mononuclear cells (PBMC) were collectedfrom the interface. DPBS was added to bring the final volume to 50 mlsand the cells were centrifuged at 350 g for 5 minutes. Pelleted cellswere resuspended in DPBS and counted.

7.1.3 Dendritic Cells

For isolation of dendritic cells, T75 culture flasks (Costar) wereseeded with 10⁸ freshly isolated PBMC in a total volume of 30 mls AIM Vmedia (Invitrogen). Excess PBMC were frozen at −80° C. in 90% fetal calfserum (FCS), 10% DMSO at 5×10⁷ cells/mL. T75 flasks were incubated at37° C. in 5% CO₂ for 2 hours. Nonadherent cells were removed, and theadherent monolayer was washed with DPBS. To differentiate dendriticcells from monocytes, 30 mls of AIM V media containing 800 units/mL ofGM-CSF (R and D Systems) and 500 units/mL IL-4 (R and D Systems) wasadded. Flasks were incubated for 5 days. On day 5 IL-1α (Endogen) andTNFα (Endogen) were added to 50 pg/mL and 0.2 ng/mL. Flasks wereincubated two more days. On day 7, dendritic cells were collected by theaddition of 3 mls of 100 mM EDTA containing 0.5 to 1.0 mg Mitomycin C(Sigma-Aldrich) for a final concentration of 10 mM EDTA and 16.5 to 33μg/mL Mitomycin C. Flasks were incubated an additional hour at 37° C.and 5% CO₂. Dendritic cells were collected, and washed in AIM V media2-3 times.

7.1.4 Cell Culture

On day 7, previously frozen autologous PBMC were thawed quickly in a 37°C. water bath. Cells were immediately diluted into DPBS or AIM V mediaand centrifuged at 350 g for 5 minutes. CD4⁺ cells were enriched bynegative selection using magnetic beads (Easy-Sep CD4⁺ kit, Stem CellTechnologies). Autologous CD4⁺ T cells and dendritic cells werecocultured at 2×10⁵ CD4⁺ T cells per 2×10⁴ dendritic cells per well in96 well round bottomed plates (Costar 9077). Peptides were added atapproximately 5 μg/mL. Control wells contained the DMSO (Sigma) vehiclealone at 0.25% v:v. Positive control wells contained DMSO at 0.25% andtetanus toxoid (List Biologicals or CalBioChem) at 1 μg/mL. Cultureswere incubated for 5 days. On day 5, 0.25 μCi per well of tritiatedthymidine (Amersham or GE Healthcare) was added. Cultures were harvestedon day 6 to filtermats using a Packard Filtermate Cell harvester.Scintillation counting was performed using a Wallac MicroBeta 1450scintillation counter (Perkin Elmer).

7.1.5 Data Analysis

Average background CPM values were calculated by averaging individualresults from 6 to 12 replicates. The CPM values of the four positivecontrol wells were averaged. Replicate or triplicate wells for eachpeptide were averaged. Stimulation index values for the positive controland the peptide wells were calculated by dividing the averageexperimental CPM values by the average control values. In order to beincluded in the dataset, a stimulation index of greater than 3.0 in thetetanus toxoid positive control wells was required. A response was notedfor any peptide resulting in a stimulation index of 2.95 or greater.Peptides were tested using peripheral blood samples from a group of 99donors. Responses to all peptides were compiled. For each peptidetested, the percentage of the donor set that responded with astimulation index of 2.95 or greater was calculated. In addition, theaverage stimulation index for all donors was also calculated.

7.2 Results

7.2.1 Identification Of CD4+ T Cell Epitopes in the Bevacizumab VH AndVL Regions

CD4⁺ T cell epitope peptides were identified by an analysis of thepercent responses to the peptides within the set of 99 donors. Theaverage percent response and standard deviation were calculated for allpeptides tested describing the bevacizumab heavy chain and light chain Vregions. A response rate greater than or equal to the average backgroundresponse plus three standard deviations was considered a potential CD4⁺T cell epitope. For the bevacizumab light chain V region, 32 peptideswere tested (FIG. 7) which resulted in an average background percentresponse of 2.1±2.7%. Three standard deviations above background wasdetermined to be 10.2%. One peptide at position 13 (Q40-T54) displayedthis level of response in the bevacizumab light chain peptide dataset,with a response rate of 15.2% (FIG. 2A). For the bevacizumab heavy chainV region, 37 peptides were tested (FIG. 8). The average backgroundpercent response was 2.8±3.1%. Three standard deviations abovebackground was 12.1%. One peptide within the bevacizumab heavy chaindataset, #18 (N52-R56), achieved a percent response of 16.2% (FIG. 3A).A second peptide at position #30 in the heavy chain dataset achieved aresponse rate of 9.1%, and was considered an epitope due to an increasedstimulation index (see below).

The average stimulation index was calculated for all peptides in thedataset. Light chain peptide 13 had a high average stimulation index of1.82±0.24 s.e.m. (FIG. 2B). Heavy chain peptide #18 had an averagestimulation index value of 2.16±0.35 s.e.m. (FIG. 3B). The peptide atposition #30 returned an average stimulation index of 1.45+0.18 s.e.m.(FIG. 3B) due to an elevated average stimulation index and an aboveaverage response rate. The peptide at position #30 was included whendetermining CD4⁺ T cell epitope content of this antibody V region. Allof these stimulation index values are significantly higher than theaverage stimulation index for all peptides in the two datasets(1.14±0.07 for all 69 heavy chain and light chain peptides).

These data indicate that there are three CD4+ T cell epitope regions inthe bevacizumab V regions (FIG. 9). In the VL region, an epitope isfound at peptide position 13 that encompasses framework 2 and two aminoacids from CDR2. The sequence contains a murine back-mutation (V46)inserted into the sequence during humanization. In FIG. 9, theCDR-derived amino acids are underlined. In the heavy chain, two epitopepeptide regions were identified. The stronger epitope at position #18encompasses all of CDR2. The second epitope peptide region contains bothframework 3 and CDR3 amino acids.

7.2.2 Reduced Immunogenicity Variants of Bevacizumab Variant Antibodies

Bevacizumab was subjected to mutational analysis (see Example 2 below).Based on antigen-binding studies performed in conjunction with themutational analysis, a set of candidate amino acid substitutions withinthe CDR-H2 and CDR-H3 region were identified that did not significantlyreduce the affinity of the antibody to VEGF (FIG. 10). These amino acidsubstitutions were tested singly and in combination to identify variantsof bevacizumab with reduced immunogenicity as compared to the wild typeantibody.

8. EXAMPLE 2 Identification of Variants of Bevacizumab with IncreasedAffinity to VEGF

The bevacizumab antibody was subjected to comprehensive mutationalanalysis to identify mutants that had increased affinity to VEGF ascompared to bevacizumab. The increased affinity of candidate highaffinity mutants to VEGF as compared to bevacizumab was analyzed byBIAcore to confirm their binding characteristics.

8.1 Materials & Methods

8.1.1 BIAcore

Fifteen variant bevacizumab VH region constructs were cloned along withthe unmodified VL region into a human IgG₁-containing plasmid, expressedin 293T/17 cell lines by transient transfection, and antibodies purifiedby Protein A or Protein G affinity. The affinity of the antibodies forVEGF (R&D systems, Minneapolis, Minn.) was determined by using a BIAcore2000 and 3000 surface plasmon resonance system (BIAcore, GE Healthcare,Piscataway, N.J.). Polyclonal goat anti-human Fc antibody (JacksonImmunoresearch) was first immobilized to the biosensor surface usingstandard BIAcore amine coupling reagents(N-ethyl-N′-dimethylamino-propylcarbodiimide, EDC; N-hydroxysuccinimide,NHS; and ethanolamine HCl, pH 8.5), followed by the capture of anti-VEGFantibodies (bevacizumab and bevacizumab variants) on parallel surfacesat a low flow rate of 5 μL/min. RL was kept low to minimize avidity dueto the dimeric nature of VEGF. No capture of the antibody was made onthe reference surface to serve as a negative control. Subsequently, VEGFwas injected to all flow cells at a flow rate of 50 μL/min for twominutes to monitor association followed by a 25-minute flow of HBS-Prunning buffer (10 mM HEPES, 150 mM sodium chloride, 0.005% P-20, pH7.4) to monitor the dissociation phase. At each cycle, VEGF, in 6different concentrations of VEGF ranging between 0 nM and 512 nM and atfour-fold increments, was injected over the surface. The surface wasregenerated with 1.5% H₃PO₄ at a flow rate of 100 μL/min in two briefpulses at the end of each cycle. Binding data were fit to the 1:1Langmuir model to extract binding constants from the BIAevaluatesoftware. Double referencing was applied in each analysis to eliminatebackground responses from the reference surface and buffer only control.All the binding kinetics data were analyzed at least three separatedeterminations.

8.2 Results

Results are displayed as absolute numbers and as fold improvement overwild-type. Almost all the variants listed have improved association(k_(on)) and dissociation (k_(off)) rates when compared to bevacizumabor wild-type (FIG. 11). The final affinity values for the variants werein the 0.1 nM range and reach as low as 0.08 nM for the variantcorresponding to SEQ ID NO:82. These values contrast to bevacizumabwhich has a measured affinity in these experiments of 1.9 nM.

FIGS. 12 and 13 show additional heavy chain variants that preliminarybinding studies show have a greater affinity to VEGF than bevacizumab(data not shown). FIG. 14 shows heavy chain variants that preliminarystudies indicate have an affinity to VEGF similar to that of bevacizumab(data not shown). FIG. 15 shows light chain variants that thatpreliminary studies indicate have an affinity to VEGF similar to that ofbevacizumab (data not shown).

9. EXAMPLE 3 Selection of Deimmunized Variant Peptides

Variant peptides corresponding to the immunogenic regions of bevacizumab(see Example 1) were generated (FIGS. 18-20). The variant peptides wereselected on the basis of comprehensive mutational analysis described inExample 2, in which CDR modifications were identified that did notsubstantially reduce the binding affinity of bevacizumab to VEGF.

A total of 77 peptides were synthesized and tested based on theantigen-binding studies, including two syntheses of each of the parent15-mer peptides. A total of 93 donors were tested with the parent andvariant peptides utilizing the method described in Section 7.1 and theresults are shown in FIGS. 4A-4C. In particular, FIGS. 4A-4C show CD4+ Tcell responses to mutant bevacizumab epitope peptides. Average responsesto the unmodified parent epitope sequences are indicated with openmarks. Large circles indicate selected peptides referred to in FIG. 21(see below). FIG. 4A shows VH CDR2 peptides; FIG. 4B shows VH CDR3peptides; and FIG. 4C shows VL CDR2 peptides. Immunogenicity data forselected peptides are shown in FIG. 21.

For the heavy chain variable region CDR2 peptides, the average percentresponse to the parent peptides in this study was 5.38% and 6.45%. Threemutant peptides demonstrated a reduced overall response rate and averagestimulation index as compared to the parent peptides.

The parent peptide response rates for the heavy chain variable regionCDR3 epitope peptides in this study were 7.53% and 6.45%. A singlemutant peptide sequence was found that demonstrated reduced overallresponses as compared to the parent peptide.

Finally, the light chain variable region CDR2 peptide parent responserates in this study were 25.8% and 15%. One mutant peptide wasidentified that demonstrated a reduced overall immunogenicity ascompared to the parent peptide.

To demonstrate that the deimmunizing mutations maintained affinity toVEGF as compared to bevacizumab, flow cytometry was used to compare thebinding properties of variant antibodies incorporating mutations in themodified epitope peptides (either as single or double amino-acidmodifications and bevacizumab). Several deimmunizing mutations hadcomparable or increased affinity to VEGF as compared to bevacizumab.

In one study, transiently transfected 293c18 cells expressingsurface-bound forms of the bevacizumab variants were stained withAlexa647-conjugated rHuVEGF (Invitrogen Cat #PHG0143) at 3 nM andgoat-anti-human-kappa-RPE (Southern Biotech Cat#2063-09) at a 1:400dilution. Data were gathered by way of flow cytometry using aDakoCytomation CyAn ADP flow cytometer and was analyzed using Treestar'sFloJo analysis program. The mean fluorescence intensities (MFI) measuredin this work are set forth in FIG. 22.

In another study, the EC₅₀ of bevacizumab and variant antibody bindingto VEGF was measured. Antibody titration plots were generated usingbevacizumab and its variants with Alexas647-conjugated rHu VEGF asdescribed above, with the VEGF serially diluted two-fold from 5 μM to0.01 μM. The EC₅₀ values are shown in FIG. 23.

10. SPECIFIC EMBODIMENTS, CITATION OF REFERENCES

All publications, patents, patent applications and other documents citedin this application are hereby incorporated by reference in theirentireties for all purposes to the same extent as if each individualpublication, patent, patent application or other document wereindividually indicated to be incorporated by reference for all purposes.While various specific embodiments have been illustrated and described,it will be appreciated that various changes can be made withoutdeparting from the spirit and scope of the invention(s).

What is claimed is:
 1. A monoclonal antibody or a binding fragmentthereof which: (a) specifically binds to human VEGF; (b) comprises aheavy chain amino acid sequence having at least 95% sequence identity toSEQ ID NO:1 and a light chain amino acid sequence having at least 95%sequence identity to SEQ ID NO:2; and (c) has at least one amino acidsubstitution or combination of amino acid substitutions selected from:(i) K64S in CDR-H2; (ii) H97E and Y98F in CDR-H3; (iii) N31F in CDR-H1,H97D in CDR-H3, Y99D in CDR-H3, and S100aG in CDR-H3; (iv) N31F inCDR-H1, H97P in CDR-H3, Y99D in CDR-H3, and S100aG in CDR-H3; (v) N31Fin CDR-H1, H97P in CDR-H3, and Y99E in CDR-H3; (vi) N31F in CDR-H1, H97Ein CDR-H3, and Y99E in CDR-H3; (vii) N31F in CDR-H1, H97D in CDR-H3, andY99E in CDR-H3; (viii) N31F in CDR-H1, H97E in CDR-H3, Y99D in CDR-H3,and S100aG in CDR-H3; (ix) N31F in CDR-H1, Y99D in CDR-H3, and S100aG inCDR-H3; (x) N31F in CDR-H1, H97P in CDR-H3, and Y99D in CDR-H3; (xi)N31F in CDR-H1, H97D in CDR-H3, and S100aG in CDR-H3; (xii) N31F inCDR-H1 and S100aG in CDR-H3; (xiii) N31F in CDR-H1, H97P in CDR-H3, andS100aG in CDR-H3; and (xiv) N31F in CDR-H1; wherein the substitutionsoccur at positions corresponding to Kabat numbering in the heavy chainof SEQ ID NO: 1 wherein the antibody or binding fragment thereof reducedimmunogenicity or increased affinity to VEGF as compared to bevacizumabor ranibizumab.
 2. The monoclonal antibody or binding fragment of claim1, wherein the heavy chain amino acid sequence has at least 98% sequenceidentity to SEQ ID NO:1 and the light chain amino acid sequence has atleast 98% sequence identity to SEQ ID NO:2.
 3. The monoclonal antibodyor binding fragment of claim 1, wherein the heavy chain amino acidsequence has at least 99% sequence identity to SEQ ID NO:1 and the lightchain amino acid sequence has at least 99% sequence identity to SEQ IDNO:2.
 4. The monoclonal antibody or binding thereof fragment of claim 1,wherein said amino acid substitution includes the substitution K64S. 5.The monoclonal antibody or binding thereof fragment of claim 1, whereinsaid amino acid substitution includes the substitution N31F.
 6. Themonoclonal antibody or binding thereof fragment of claim 1, wherein saidamino acid substitutions include the substitutions H97E and Y98F.
 7. Themonoclonal antibody or binding fragment thereof of claim 1, wherein theantibody or binding fragment thereof is human or humanized antibody. 8.The monoclonal antibody or binding fragment thereof of claim 1, whereinthe antibody is an IgG.
 9. The monoclonal antibody or binding fragmentthereof of claim 1, wherein the antibody or binding fragment thereof hasthe heavy chain framework sequences of the V_(H) sequence of SEQ ID NO:1and the light chain framework sequences of the V_(L) sequence of SEQ IDNO:2.
 10. The monoclonal antibody or binding fragment thereof of claim1, wherein the antibody or binding fragment thereof has the heavy chainframework sequences of the V_(H) sequence of SEQ ID NO:9 and the lightchain framework sequences of the V_(L) sequence of SEQ ID NO:10.
 11. Anantibody-drug conjugate comprising the anti-VEGF antibody or anti-VEGFbinding fragment according to claim 1.